WO2019134630A1 - 一种Cas蛋白体系分离DNA的方法 - Google Patents

一种Cas蛋白体系分离DNA的方法 Download PDF

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WO2019134630A1
WO2019134630A1 PCT/CN2018/125973 CN2018125973W WO2019134630A1 WO 2019134630 A1 WO2019134630 A1 WO 2019134630A1 CN 2018125973 W CN2018125973 W CN 2018125973W WO 2019134630 A1 WO2019134630 A1 WO 2019134630A1
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dna
protein
cas protein
cas
solid support
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PCT/CN2018/125973
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French (fr)
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吴尧
王兴兴
李秋实
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苏州克睿基因生物科技有限公司
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Priority to US16/960,037 priority Critical patent/US20210095269A1/en
Priority to CN201880079130.5A priority patent/CN111448315B/zh
Priority to CN202311149195.1A priority patent/CN116970601A/zh
Priority to EP18898516.2A priority patent/EP3722422B1/en
Publication of WO2019134630A1 publication Critical patent/WO2019134630A1/zh

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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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Definitions

  • the present invention relates to a method of isolating DNA, and more particularly to a method for isolating DNA from a solution using Cas protein.
  • DNA deoxyribonucleic acid
  • the extracellular DNA found in each sample is protected by nucleases (for example, they are secreted in the form of a protein lipid complex, often bound to proteins, or contained in vesicles) and are therefore not easily degraded. .
  • nucleases for example, they are secreted in the form of a protein lipid complex, often bound to proteins, or contained in vesicles
  • extracellular DNA is often much higher than normal, so it is used in disease screening, diagnosis, prognosis, disease progression monitoring, and even the identification of therapeutic targets.
  • Other aspects have great application prospects.
  • maternal blood is often accompanied by a considerable amount of fetal DNA, which can be used for a variety of tests, such as gender identification, testing and evaluation of chromosomal abnormalities, as well as monitoring pregnancy-related complications. It is also because of its strong clinical diagnostic relevance that free DNA has great potential for non-invasive diagnosis and prognosis, and can be used for many other diseases such as non-invasive prenatal genetic testing, tumor detection, and transplantation medicine
  • CRISPR clustered regular short palindrome repeat
  • Cas protein CRISPR binding protein
  • the RNA component is a double-stranded guide RNA structure consisting of a CRISPR RNA (crRNA) and a trans-activated crRNA (tracrRNA), directing the Cas protein to cleave a DNA site complementary to the crRNA sequence (Jinek et al. Science 2012.337: 816-821).
  • crRNA CRISPR RNA
  • tracrRNA trans-activated crRNA
  • the researchers used engineering methods to link the two components of the double-stranded guide RNA (crRNA and tracrRNA) into chimeric single-stranded guide RNA (sgRNA). This reflects the powerful gene recognition and gene editing capabilities of the CRISPR/Cas9 system, but the nature of the Cas protein itself has not been studied in depth. To date, no researchers have discovered the potential of using Cas protein as a nuclease alone and its variants to recognize and bind DNA.
  • the Cas protein system is a Cas fusion protein formed by a Cas protein and at least one ligation sequence.
  • step b) adds a solid support to which an affinity molecule is coupled to the solution, and is further enriched by centrifugation or magnetically enriched.
  • Solid matter is selected from the group consisting of at least one of a gel material, a magnetic material, a cellulose material, a silica gel material, a glass material, and a man-made polymer. Solid matter.
  • linker sequence and the affinity molecule are selected from the group consisting of a combination of a ligand and a substrate, an enzyme and a substrate, Antigens and antibodies, biotin and avidin.
  • the antigen-antibody is selected from the group consisting of A protein or a fragment thereof which retains the function of binding to an Fc region and an immunoglobulin or an Fc thereof Or Fab fragment, G protein or a fragment thereof which retains the functionality of the Fc region and immunoglobulin or its Fc or Fab fragment, histidine tag and anti-histidine tag, polyhistidine tag and anti-polyhistidine Labels and FLAG tags with anti-FLAG tags.
  • biotin and avidin are selected from one of the group consisting of biotin and avidin or streptavidin, and biotin linked thereto.
  • reaction time of the step a) is from 5 minutes to 6 hours, preferably from 15 minutes to 1 hour.
  • reaction temperature of the step a) is 20-37 ° C, preferably the reaction temperature is 30-37 ° C.
  • step b) The method according to any one of the preceding aspects, wherein the step b), the reaction of adding a solid support coupled with an affinity molecule to the solution
  • the time is from 30 minutes to overnight, preferably the reaction time is 1-2 hours.
  • reaction temperature of the step b) is 4 ° C to 37 ° C, preferably the reaction temperature is 20-30 ° C.
  • step c) is to isolate the DNA by elution.
  • a kit for isolating DNA from a solution comprising a component a) a Cas protein system, and a component b) a DNA eluate.
  • the kit according to the invention (28), wherein the component a) Cas protein system is a Cas protein solution system coupled with a solid support, or a Cas fusion protein and an affinity molecule coupled thereto.
  • kits according to the invention wherein the component a) the Cas protein system consists of two independent systems, i) the Cas protein and the Cas fusion protein formed by at least one ligation sequence, and ii) A solid support coupled to the affinity molecule.
  • DNA eluate may be the following liquid: 0.5-10 mg/ml proteinase K solution, 1-5 M NaCl or KCl salt solution, having A base eluate of 0-0.3 M Tris and 0-0.5 M NaCl at pH 11-12.5.
  • the solid support is selected from the group consisting of a gel material, a magnetic material, a cellulose material, a silica gel material, a glass material, and a solid material prepared by a man-made polymer. At least one of them.
  • kits according to the invention (34), wherein the solid support is a magnetic bead, a gel bead, and/or a silica gel bead.
  • the linker sequence and the affinity molecule are selected from the group consisting of one of a combination of a ligand and a receptor: an enzyme and a substrate, an antigen and Antibodies, biotin and avidin.
  • the antigen-antibody is selected from the group consisting of A protein or a fragment thereof which retains the function of binding to an Fc region and an immunoglobulin or Fc or Fab fragment, G protein or a fragment thereof which retains the functionality of the Fc region and immunoglobulin or its Fc or Fab fragment, histidine tag and anti-histidine tag, polyhistidine tag and anti-multi-group ammonia Acid label and FLAG label with anti-FLAG label.
  • biotin and avidin are selected from the group consisting of biotin and avidin or streptavidin, and a living organism A biotin receptor protein with avidin or streptavidin, Strep-tag and avidin or streptavidin.
  • the kit according to the invention wherein the Cas protein is a natural Cas protein, a Cas protein which has lost gRNA binding ability after mutation, or a Cas protein inactivated by a nuclease after mutation.
  • kits according to the invention wherein the Cas9 protein is a dCas9 protein (a nuclease-inactivated Cas9 protein after mutation).
  • the extraction method of the invention eliminates the cumbersome sample processing process, reduces the sample usage requirement, and greatly improves the efficiency of extracting free DNA.
  • Figure 1 Expression vector pSMART-his-S.P.dCas9-FLAG containing the his-S.P.dCas9-FLAG gene fragment
  • FIG. 2 Electrophoresis band of 486 bp free DNA (SEQ ID NO. 8) binding to dCas protein.
  • Lane 1 is the band of free DNA itself
  • Lane 2 is the case after the binding of dCas9 and free DNA (SEQ ID NO. 8)
  • the clustered regular interval palindrome repeat (CRISPR) and the CRISPR binding protein (Cas protein) constitute a powerful nuclease system.
  • the CAS protein can be divided into four different functional modules: target recognition module (interval acquisition); expression module (crRNA processing and target binding); interference module (target cutting); and auxiliary modules (regulatory and other CRISPR related functions).
  • target recognition module interval acquisition
  • expression module crRNA processing and target binding
  • interference module target cutting
  • auxiliary modules auxiliary modules
  • the core Cas protein (Cas1-Cas10) has accumulated a large amount of structural and functional information, which allows them to be classified into these modules.
  • the CRISPR-associated endonuclease Cas protein can target specific genomic sequences via single-stranded guide RNA (sgRNA).
  • the protein comprises a bilobal structure consisting of a target recognition module and a nuclease module (interference module), which is in a positively charged groove at the interface.
  • a nuclease module interference module
  • the nuclease region comprises HNH and RuvC nuclease domains which are suitable for cleavage of complementary and non-complementary strands of the target DNA, respectively.
  • the nuclease region also contains a carboxy terminal domain responsible for interaction with the protospacer neighboring motif (PAM).
  • PAM protospacer neighboring motif
  • the Cas protein itself is a nucleic acid binding system in the absence of RNA mediated.
  • the Cas protein recognizes and binds to almost all DNA sequences in the absence of RNA and does not cleave the bound DNA sequence.
  • the Cas protein of the present invention is a Cas protein having DNA binding function, such as a natural Cas protein, a mutated Cas protein, a Cas protein which has lost gRNA binding ability after mutation, and a nuclease-inactivated Cas protein (dead Cas) after mutation. , dCas).
  • Cas proteins include: Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Cas12, Cas13, Cas14, Csy1, Csy2, Csy3 , Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3 , Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologous proteins in different species, mutant proteins inactivated by endonucleases, or modified forms thereof.
  • the Cas protein of the invention is a Cas9 protein, more preferably a nuclease-inactivated Cas9 protein (dead Cas9, dCas9)
  • Cas9 also known as Csn1 or Csx12, is a giant protein involved in both crRNA biosynthesis and in the destruction of invading DNA.
  • S. thermophiles Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and Streptococcus pyogenes Cas9 is described in (S. Pyogenes) (Deltcheva, Chylinski et al. 2011).
  • the Cas9 of the present invention includes, but is not limited to, the following types: Streptococcus pyogenes Cas9 protein, the amino acid sequence of which is described in SwissProt database accession number Q99ZW2; Neisseria meningitides Cas9 protein, the amino acid sequence of which is described in the UniProt database. No. A1IQ68; Streptococcus thermophilus Cas9 protein, the amino acid sequence of which is shown in UniProt database number Q03LF7; Staphylococcus aureus Cas9 protein, the amino acid sequence of which is shown in UniProt database number J7RUA5.
  • the Cas protein acting on the DNA double strand generally has two endonuclease active sites. If one or only one site is mutated or deleted, the enzyme activity of the site is invalidated, and a Nickase that cleaves the DNA single strand, such as Cas9, can be obtained. -nickase is widely used due to its high fidelity and the ability to cut single strands of DNA.
  • the sequence of its protein is also truncated by protein engineering research to form a Cas protein system, for example, the Split-SpCas9 system contains a truncation.
  • the Split-SpCas9 system contains a truncation.
  • the Cas protein is a fusion protein or protein complex comprising the above protein or a mutant thereof, including, but not limited to, a variant that joins other amino acid sequences on Cas. Fusion of other functional proteins based on the above Cas protein or its variant can increase the specificity and effectiveness of Cas protein function, and can also produce effects other than cleavage on the genome. For example, fusion of FokI on Cas or dCas can increase the specificity of Cas9 protein for genomic cleavage, because FokI only has dimerization to have cleavage activity, which requires a pair of recognition regions to complete the cleavage and reduce the off-target rate.
  • FokI is ligated on a Cas protein that truncates a portion of the functional domain but retains its DNA binding ability (Ma et al. ACS Synth. Biol., 2018, 7(4), pp 978-985).
  • a modified enzyme such as deaminase, cytosine deaminase, adenine deaminase
  • dCas9 can be fused to a protein domain that regulates gene expression, which can effectively regulate the expression of target genes.
  • dCas9 which is a transcriptional activator such as VP64, VPR
  • a transcriptional repressor such as SRDX
  • dSpCas9-Tet1 and dSpCas9-Dnmt3a can be used to modify the epigenetic state and regulate the methylation status of the endogenous gene promoter to regulate protein expression.
  • the Cas protein having a DNA-binding function may be a native Cas protein, or a mutation made outside the DNA-binding domain of the Cas protein, or a Cas protein obtained after mutation in the DNA-binding domain.
  • the detection of whether the Cas protein has a DNA-binding function can be discriminated by gel electrophoresis comparing the position of the gel of the bound protein and the DNA of the unbound protein (see Example 1 for the detection method).
  • Nuclease-inactivated mutant Cas protein is a variant obtained by mutation of Cas protein, and its endonuclease activity is inactivated or substantially inactivated, which causes Cas protein to lose or substantially lose endonuclease activity, and further The target sequence could not be cut.
  • Non-limiting examples of the aforementioned Cas proteins can be engineered into dCas proteins by inactivation of the endonuclease mutations, including insertions, deletions or substitutions of one or more amino acid residues.
  • Cas9 mutations can cause the Cas9 protein to lose or substantially lose endonuclease activity, thereby failing to cleave the target sequence.
  • Exemplary mutations that reduce or eliminate endonuclease activity for a species of Cas9, such as spCas9 include one or more of the following positions: D10, G12, G17, E762, H840, N854, N863, H982 , H983, A984, D986, or A987.
  • gRNA targeting RNA
  • the dCas protein may be a variant that is not naturally found in nature and is obtained by protein engineering or by random mutagenesis, in which the endonuclease activity is inactivated or substantially inactivated.
  • the corresponding dCas9 protein can be obtained by mutation, deletion or insertion or substitution of at least one residue in the amino acid sequence of S. pyogenes Cas9 endonuclease.
  • the dCas9 protein can be a variant of Cas9 of different species that is obtained by inactivation or substantial inactivation of the endonuclease activity, including but not limited to, Streptococcus pyogenes Cas9 protein, meningitis Neisseria meningitides Cas9 protein, Streptococcus thermophilus Cas9 protein, Staphylococcus aureus Cas9 protein, Streptococcus pneumoniae Cas9 protein, and the like.
  • the linker sequence of the present invention refers to a molecule that is directly or indirectly linked to the C-terminus or the N-terminus of the Cas protein, which ultimately forms a fusion protein with the Cas protein.
  • the affinity molecule of the present invention is a molecule coupled to a solid phase material and capable of forming a specific binding to a linker sequence on the aforementioned Cas protein.
  • the linker sequences and affinity molecules of the invention are each a receptor and ligand relationship. Any protein linking sequence can be used in the practice of the present invention as long as it can specifically bind to an affinity molecule.
  • the ligation sequence and the affinity molecule are selected from one of the following combinations of ligand receptors: an enzyme and a substrate, an antigen-antibody, biotin and avidin. Wherein the enzyme and substrate are selected from the group consisting of glutathione transferase and glutathione; the antigen-antibody is selected from one of the group consisting of protein A or retained therein.
  • biotin and avidin are selected from one of the group consisting of biotin ( Biotin) with avidin (Avidin, UniProt database number P02701) or streptavidin (UniProt database code P22629), biotin-binding biotin receptor protein (such as AviTag, amino acid sequence: GLNDIFEAQKIEWHE) and pro Avidin or streptavidin, Strep-tag (amino acid sequence: WSHPQFEK) and avidin or streptavidin.
  • biotin and avidin are selected from one of the group consisting of biotin ( Biotin) with avidin (Avidin, UniProt database number P02701) or streptavidin (UniProt database code P22629), biotin-binding biotin receptor protein (such as AviTag, amino acid sequence: GLNDIFEAQKIEWHE) and pro Avidin or streptavidin, Strep-tag (amino acid sequence: WSHPQFEK) and avidin or str
  • the linker sequence and the affinity molecule specifically bind by affinity and are interchangeable.
  • the linker sequence is a receptor
  • the affinity molecule is a ligand that specifically binds to the receptor
  • the linker sequence may also be the ligand
  • the affinity molecule is a receptor that specifically binds to the ligand.
  • the linker sequence is streptavidin
  • the affinity molecule is biotin; the linker sequence can also be biotin, then the affinity molecule is streptavidin.
  • the solid phase material of the present invention is a solid substance capable of forming a chemical bond with an amino acid residue, and thus can be coupled to an affinity molecule or a Cas protein by a chemical bond.
  • Suitable solid supports can be distinguished from materials by gel materials, magnetic materials, cellulosic materials, silica gel materials, glass materials, or solid polymeric polymers.
  • the gel material may be a hydrogel, an organogel, a xerogel, or a nanocomposite hydrogel;
  • the magnetic material may be a metal oxide (such as an oxide of iron, cobalt and nickel) encapsulated by a polymer material;
  • a cellulosic material It may be cellulose, cellulose acetate or nitrocellulose;
  • the silica gel material may be a silica support or an organic silica gel support;
  • the artificial high molecular polymer may be, nylon, polyester, polyethersulfone, polyolefin, polyposition 1,1-difluoroethylene and combinations thereof.
  • the solid phase support may be formally distinguished by a bead shape, a chemical film column, a glass plate, a 6-well/24-well/48-well/96-well/384
  • the Cas protein combined with DNA is eluted with a solid support such as a chemical membrane column, a glass plate, a 6-well/24-well/48-well/96-well/384-well plate, and a PCR reaction tube. Come down.
  • a solid support such as a chemical membrane column, a glass plate, a 6-well/24-well/48-well/96-well/384-well plate, and a PCR reaction tube.
  • the DNA-binding Cas protein is bound to the bead-shaped solid support, and after enrichment, such as centrifugation or magnetic enrichment, the DNA is eluted by the eluent.
  • the solid support of the present invention is preferably beaded, such as magnetic beads, gel beads and/or silica gel beads.
  • gel beads that can be used include: Anti-6X His Antibody (Agarose Gel) (Abcam), Anti- 6xHis Acceptor beads (PerkinElmer), Dynabeads TM His-Tag Isolation and Pulldown (ThermoFisher), Anti-His-tag mAb-Magnetic Beads (MBL Life science), His Tag Antibody Plate (GenScript).
  • Affinity molecules or Cas proteins can be coupled to a solid support by conventional methods.
  • the A protein and the G protein are linked to a solid support, see, for example, Hermanson et al. 1992, Immobilized Affinity Ligand Techniques, Academic Press.
  • the solid support is a reactive functional group ("activating group") such as an epoxide (epichlorohydrin), cyanide (cyanogen bromide CNBr), N,N-disuccinimidyl carbonate (DSC).
  • activating group such as an epoxide (epichlorohydrin), cyanide (cyanogen bromide CNBr), N,N-disuccinimidyl carbonate (DSC).
  • An aldehyde or an activated carboxylic acid such as N-hydroxysuccinimide (NHS) ester, carbonyl diimidazole (CDI) activated ester is activated.
  • activating groups can be attached directly to a solid support, such as CNBr, or they can be part of a "linker” or spacer molecule, typically a linear chain of carbon, oxygen and nitrogen atoms, as in the linker butanediol II.
  • a linker typically a linear chain of carbon, oxygen and nitrogen atoms, as in the linker butanediol II.
  • the activated solid support is then equilibrated with the Cas protein or affinity molecule under coupling conditions. The coupling reaction is completed and the medium is thoroughly washed.
  • the Cas fusion protein of the present invention is a fusion protein formed by adding a ligation sequence to the N-terminus or C-terminus of the Cas protein or any part of its protein sequence.
  • the fusion Cas protein of the present invention can express the Cas protein together with the ligation sequence by a genetic engineering method.
  • the Cas protein system of the present invention is a complex comprising a Cas protein for binding DNA in a solution.
  • the Cas protein system is a complex formed by the coupling of a Cas protein to a solid support by chemical covalent bonds.
  • the Cas protein system is a Cas fusion protein formed by the Cas protein with one or more ligation sequences.
  • the Cas protein system is a complex formed by binding of a Cas fusion protein to an affinity molecule coupled to a solid phase material.
  • the Cas protein forms a Cas fusion protein together with the ligation sequence.
  • the ligation sequence can be located directly or indirectly at the N-terminus or C-terminus of the Cas protein.
  • a solid phase material as a gel bead as an example, in a preferred embodiment, when a gel bead of an affinity molecule is used to bind a Cas fusion protein, for every 10 ⁇ l of the gel bead, before the addition of the gel bead
  • the initial concentration of the Cas fusion protein in the reaction solution is 0.001-20 ⁇ g/ ⁇ l, preferably 0.01-10 ⁇ g/ ⁇ l of the Cas9 fusion protein.
  • the binding of the Cas fusion protein to the solid phase material is carried out at 4 ° C to 37 ° C, preferably 4 ° C or room temperature (20-25 ° C).
  • the binding reaction solution used may be deionized water, 1x phosphate buffered saline (PBS), Tris buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4), or other salt ion concentration is lower than
  • the pH of the 150 mM, oil-free detergent and denaturant is about neutral buffer.
  • the reaction time is from 30 minutes to overnight, preferably overnight.
  • the Cas protein can also bind directly to the solid phase material to which the affinity molecule is coupled without the connexin.
  • the Cas protein of the present invention can bind to DNA in any free state.
  • the DNA can be double stranded or single stranded DNA.
  • the free DNA bound to the Cas protein can be separated into a liquid phase by the solid phase substance binding to the Cas protein.
  • the DNA that binds to the Cas protein of the present invention may be any free DNA.
  • any sequence DNA fragment obtained by PCR using the genome as a template the sequence is an artificially designed DNA fragment, free DNA after cell lysis, a genome obtained after cell lysis, and free DNA in human or animal body fluid.
  • the DNA is a DNA fragment obtained by PCR using a genome as a template, an artificially designed DNA fragment, or a free DNA in a human or animal body fluid.
  • the protein concentration in the DNA binding reaction solution is 0.001 to 20 ⁇ g/ ⁇ l. Preferably, 0.01 to 10 ⁇ g/ ⁇ l of Cas protein or a variant thereof or a Cas fusion protein.
  • the reaction is carried out at room temperature (20-25 ° C) to 37 ° C.
  • the DNA binding reaction solution may be deionized water, Phosphate buffered saline (PBS), Tris buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4), or other low salt ion concentration.
  • PBS Phosphate buffered saline
  • Tris buffer 50 mM Tris-HCl, 150 mM NaCl, pH 7.4
  • the pH is neutral buffer at 150 mM, oil-free detergent and denaturing agent.
  • Preferred is 50 mM KCl, 10 mM EDTA, 30 mM Tris-HCl, 0.2% Triton X-100, 12% glycerol.
  • the reaction time is from 5 minutes to 6 hours, and in one embodiment, the preferred time is from 5 minutes to 2 hours. In another embodiment, the preferred time is from 15 minutes to 1 hour.
  • the method for isolating free DNA of the Cas protein of the present invention can be used in three ways:
  • the Cas protein or the Cas fusion protein is first bound to the free DNA in the DNA binding reaction solution, and then the Cas protein or the Cas fusion protein is bound by the solid phase of the coupled affinity molecule in the protein binding reaction solution.
  • the Cas protein or Cas fusion protein is incubated with the free DNA in the DNA binding reaction solution under the conditions of the DNA binding reaction described above, preferably for 5 minutes to 6 hours, and in another embodiment, the preferred time is 15 minutes to 1 hour.
  • the reaction temperature is room temperature (20-25 ° C) to 37 degrees; after the end of the DNA binding reaction, the solid phase substance coupled to the affinity molecule is directly added to the solution, and the Cas protein or Cas is obtained under the conditions of protein binding reaction as described above.
  • the fusion protein is combined with a solid phase substance coupled to the affinity molecule, preferably for a reaction time of 30 minutes to overnight, and the reaction temperature is 4 ° C to 37 ° C; finally, the solid phase material is separated by centrifugation, and the supernatant is discarded; After the second, the Cas protein or its variant or Cas fusion protein and the bound DNA are eluted from the solid phase material to obtain an elution solution.
  • the eluent can be used as a solution containing 0.5-10 mg/ml proteinase K, 1 -5M NaCl or KCl salt solution, base eluent (0-0.3M Tris, 0-0.5M NaCl, pH 11-12.5), acid eluent (0-0.3M glycine HCl, pH 2.5-3.5) , Competing eluent (DYKDDDK or FLAG amino acid TBS solution, amino acid concentration can be 100 to 500 ⁇ g / ml), or PAGE gel sample solution (0.01M Tris-HCl, 10% Glycerol, 0.016% bromophenol blue).
  • the solid phase substance coupled to the affinity molecule first binds to the Cas protein or its variant or the Cas fusion protein, and then incubated with the free DNA in the DNA binding reaction solution.
  • the reaction conditions used are the same as the solution, elution conditions and method 1.
  • the solid phase substance coupled with the affinity molecule, the Cas9 protein or a variant thereof or the Cas fusion protein is simultaneously added to the DNA binding reaction solution, and the solid phase substance is separated after the incubation.
  • the solution in the method may be a solution of the above Cas protein or a variant thereof or a Cas fusion protein in combination with free DNA, preferably for a reaction time of 30 minutes to 6 hours, and a reaction temperature of room temperature (20-25 ° C) to 37 degrees.
  • the elution conditions are the same as in the first method.
  • the DNA binding activity of the Cas protein can be determined by gel electrophoresis, comparing the position of the gel strip of the Cas protein-binding DNA and the Cas protein-free DNA.
  • the following reaction conditions were employed:
  • Reaction Buffer 50 mM Tris-HCl (pH 7.4); 150 mM Sodium Chloride (NaCl)
  • the above reaction system is added to a 200 ⁇ l PCR reaction tube and uniformly mixed, and then reacted at room temperature or 37 ° C for 15 minutes to 1 hour. After the reaction was completed, a non-denaturing gel was used, and it was operated at a voltage of 80 V and a current of 400 mA for 80 minutes in a BioRad electrophoresis tank. After removing the non-denatured gel, the nucleic acid was stained with DuRed (Shanghai Shengsheng Biotechnology Co., Ltd., 10202ES76), and then a gel imager was used to take a picture (see Fig. 2). The electrophoresis band of the DNA binding to the Cas protein apparently lags behind the electrophoresis band of the DNA that does not bind to the Cas protein.
  • the free DNA in the present invention may be DNA of any sequence.
  • SEQ ID NO. 1-6 The DNA sequence of SEQ ID NO. 1-6 was synthesized by direct PCR using the forward and reverse primers (Table 2) without using any template. The primary purpose of using these six DNAs is to determine whether the protospacer adjacent motif (PAM) region will have an effect on the binding of Cas protein to DNA.
  • PAM protospacer adjacent motif
  • SEQ ID NO. 1-3 100 bp and 4-6 is 200 bp, wherein the sequences 1 and 4 do not have a corresponding PAM region (NGG) of Cas9, and each of the sequences 2 and 5 has a PAM region, and 3 and 6 There are 3 PAM areas on each sequence. Detailed results will be discussed in Example 4.
  • the DNA sequence of SEQ ID NO. 7-11 was prepared by separately designing corresponding forward and reverse primers and using 293T cell (Shanghai Institute of Biosciences, GNHu17) genome as a template PCR reaction.
  • Table 2 Upstream and downstream primers corresponding to different free DNA sequences, and annealing temperatures of respective PCR reactions.
  • Upstream primer Downstream primer Annealing temperature SEQ ID NO: 1 SEQ ID NO: 12 SEQ ID NO: 13 55 SEQ ID NO: 2 SEQ ID NO: 14 SEQ ID NO: 15 55 SEQ ID NO: 3 SEQ ID NO: 16 SEQ ID NO: 17 55 SEQ ID NO: 4 SEQ ID NO:18 SEQ ID NO: 19 58 SEQ ID NO: 5 SEQ ID NO: 20 SEQ ID NO: 21 58 SEQ ID NO: 6 SEQ ID NO: 22 SEQ ID NO: 23 58 SEQ ID NO:7 SEQ ID NO:24 SEQ ID NO: 25 68 SEQ ID NO:8 SEQ ID NO:26 SEQ ID NO:27 60 SEQ ID NO: 9 SEQ ID NO:28 SEQ ID NO:29 60 SEQ ID NO: 10 SEQ ID NO: 30 SEQ ID NO: 31 59 SEQ ID NO: 11 SEQ ID NO:32 SEQ ID NO:33 66
  • the PCR reaction of DNA of SEQ ID NO. 1-6 was carried out using the two upstream and downstream primers described above and the Q5 hot-start ultra-fidelity 2X Master Mix kit (New England Biolabs, M0494S) with a final concentration of 0.5 ⁇ M per primer.
  • Add 10 ⁇ l of Q5 2X Master Mix add water to a total volume of 50 ⁇ l, and perform PCR according to the manufacturer's instructions.
  • the DNA sequence of SEQ ID NO. 7-11 was additionally added to 1 ⁇ l of the cell genome extract based on the above reaction system, and finally made up to 50 ⁇ l.
  • pSMART-his-S.P.dCas9-FLAG The following is a vector circular schematic, the his-SPdCas9-FLAG gene fragment (SEQ ID No: 34) is integrated into the pSMART vector by EcoRI and BamHI restriction endonucleases (see Figure 1); if other ligation sequences are used, Replace the FLAG (FLAG sequence: GATTACAAGGATGACGATGACAAG) in the vector with the corresponding sequence, such as AVI-Tag, and the other steps for expressing the purified protein are the same:
  • Instrument consumables Shaker, LB medium (labor, A507002)
  • Step: The protein solution was added to a concentrating tube, centrifuged at 4 ° C, 5000 rpm, 40 minutes, the concentrating tube was taken out, the penetrating solution was removed, and 15 ml of protein preservation solution (20 mM Hepes, pH 7.5; 150 mM KCl; 1% sucrose; 30%) was added. Glycerol; 1 mM dithiothreitol (DTT)), centrifuged at 4 ° C for 40 minutes; the above steps were repeated 3 times to obtain a final his-SPd Cas9-FLAG protein solution, which was stored at -80 ° C after dispensing.
  • the CRISPR/Cas system recognizes and cleaves specific DNA sequences based on the PAM region (sequence: NGG, N can be A/T/C/G) and RNA guidance.
  • sequence: NGG, N can be A/T/C/G RNA guidance.
  • RNA guidance RNA guidance.
  • SEQ ID NO. a DNA sequence of SEQ ID NO. Among them, 1-3 is 100 bp, and 4-6 is 200 bp. There are no corresponding PAM regions (NGG) of Cas9 on sequences 1 and 4, and one PAM region on each of sequences 2 and 5, and each of sequences 3 and 6 There are 3 PAM areas.
  • the dCas9 protein used in the reaction was identical to that of Examples 5, 6 and 7, ie, the purified FLAG-dCas9 protein was produced in Example 3.
  • the reaction conditions are as follows:
  • Reaction Buffer 50 mM Tris-HCl (pH 7.4); 150 mM Sodium Chloride (NaCl)
  • the above reaction system is added to a 200 ⁇ l PCR reaction tube and uniformly mixed, and then reacted at room temperature or 37 ° C for 15 minutes to 1 hour. After the reaction was completed, a non-denaturing gel was used, and it was operated at a voltage of 80 V and a current of 400 mA for 80 minutes in a BioRad electrophoresis tank. After removing the non-denatured gel, the nucleic acid was stained with DuRed (Shanghai Shengsheng Biotechnology Co., Ltd., 10202ES76), and then photographed using a gel imager (Fig. 2).
  • Figure 2 shows a binding electrophoresis band of free DNA (SEQ ID NO. 7) of about 500 bp and dCas protein.
  • Lane 1 is the band of free DNA itself and belongs to the negative control; while lane 2 describes the situation after the binding of dCas9 and free DNA (SEQ ID NO. 7).
  • the band of free DNA was clear and complete, and the electrophoresis was successful.
  • the second lane due to the combination of free DNA and dCas9, DNA could not be electrophoresed normally. Almost all DNA remained in the lane well, so in the corresponding place There are no obvious bands.
  • E1 binding ratio of dCas9 to DNA
  • This formula uses the brightness of lane 1 as the reference for total DNA.
  • the corresponding strip brightness in lane 2 is the amount of DNA not bound to dCas9, so the molecular calculation is the amount of DNA bound to dCas9 protein. Dividing the binding efficiency of dCas9 and DNA by the total amount of DNA, the binding efficiency of dCas9 protein and DNA was calculated to be 97.4%.
  • Table 3 summarizes the dCas9-DNA binding ratios for different free DNA lengths. The results show that dCas9 can bind to the above 11 DNAs without distinction and high efficiency, demonstrating good potential for isolating nucleic acids from solution. .
  • Example 5 FLAG-conjugated dCas9 protein binds to free solid DNA in solution and binds to solid phase beads and separates it
  • DNA (SEQ ID NO. 7, 8, 9, 11 representing the length of different free DNA): 0.25 ⁇ g per DNA
  • the above reaction system is added to a 1.5 ml PCR reaction tube and mixed uniformly, and then reacted at room temperature or 37 ° C for 15 minutes to 1 hour.
  • the mixture was centrifuged at 5000 RPM for 30 seconds, the supernatant was discarded, 150 ⁇ l of the buffer (BA) of Example 4 was added, mixed uniformly, and centrifuged at 5000 RPM for 30 seconds, and the supernatant was discarded, and the above procedure was repeated once.
  • 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403) was added to the gel beads, and 20 ⁇ l of BA was added. After mixing uniformly, the mixture was heated in a 55 ° C water bath for 30 minutes, centrifuged at 5000 RPM for 30 seconds, and the supernatant was taken to obtain isolated free DNA.
  • Example 6 FLAG-conjugated dCas9 protein is bound to solid phase beads, and the free DNA is bound in solution and separated.
  • Second step 15 ⁇ l of Anti-FLAG affinity gel beads (GenScript, L00439-1) was added to a 1.5 ml centrifuge tube, 150 ⁇ l of the buffer (BA) of Example 4 was added, mixed uniformly, and centrifuged at 5000 RPM. Seconds, discard the supernatant and repeat the above steps once. After discarding the supernatant, 500 ⁇ l of BA was added to the gel beads, and the gel beads were uniformly mixed. Then, 1 ⁇ l of a 10 ⁇ g/ ⁇ l dCas9 protein solution was added to the mixture, and the mixture was inverted and mixed at room temperature for 1 hour.
  • the second step after the reaction is finished, centrifuge at 5000 RPM for 30 seconds, discard the supernatant, add 150 ⁇ l of the buffer (BA) in Example 4, mix well, centrifuge at 5000 RPM for 30 seconds, discard the supernatant, and repeat the above steps. once.
  • the washed gel beads were separately added to 4 tubes and 1 ml of free DNA solutions containing different lengths (the free DNA used SEQ ID NOS. 7, 8, 9, and 11, representing the lengths of different free DNAs of 957, 486, 166, and 2655 bp, respectively. ), mix and react at room temperature or 37 ° C for 15 minutes to 1 hour.
  • the third step after the reaction is completed, centrifuge at 5000 RPM for 30 seconds, discard the supernatant, add 150 ⁇ l of the buffer (BA) in Example 4, mix well, centrifuge at 5000 RPM for 30 seconds, discard the supernatant, and repeat the above steps. once. After discarding the supernatant, 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403) was added to the gel beads, and 20 ⁇ l of BA was added. After mixing uniformly, the mixture was heated in a 55 ° C water bath for 30 minutes, centrifuged at 5000 RPM for 30 seconds, and the supernatant was taken to obtain isolated free DNA.
  • BA buffer
  • RT403 proteinase K
  • Positive control the same amount of DNA (free DNA using SEQ ID NOS. 7, 8, 9, 11 representing the length of different free DNA, 957, 486, 166 and 2655 bp, respectively) dissolved in 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403 ) and add 20 ⁇ l of BA.
  • the DNA captured by the present invention, the DNA captured by the Kaijie kit, and the positive control were used for quantitative analysis of DNA using the Agilent 2100 High Sensitivity DNA Analysis Kit (Cat. 5067-4626), and the amount of DNA captured and grasped was obtained.
  • the percentage results are summarized in Table 4.
  • the extraction efficiency is the ratio of the amount of DNA captured by the present invention or the Kaijie kit to the amount of positive control DNA in Table 4 (i.e., the percentage of DNA grabbed).
  • the free DNA extraction efficiency of the invention is close to 80%, and The Circulating Nucleic Acid Kit is less than 50%, so the present invention increases the extraction efficiency of free DNA by about 60%.
  • Example 7 Binding of Cpf1 (Cas12a) protein with His-Tag to solid phase beads, combining free DNA in solution and separating it
  • Step 1 Cpf1 (Cas12a) protein with His-Tag (IDT, AsCas12a (Cpf1) V3, Cat.No.1081068) with His-Tag separating beads (Dynabeads TM His-Tag Isolation and Pulldown, Cat.No.10103D) connection.
  • IDT AsCas12a
  • Cpf1 His-Tag
  • mix well place the tube in the sample well of the magnetic separator (Beaver, Cat. No. 60201) and let stand for two minutes.
  • Step 2 After the reaction is completed, the centrifuge tube containing the reaction solution and Dynabeads is placed in the corresponding sample well of the magnetic separator, allowed to stand for two minutes, the supernatant is discarded, and 150 ⁇ l of the buffer (BA) in Example 4 is added. Place the centrifuge tube in the corresponding sample well of the magnetic separator, let stand for two minutes, discard the supernatant, and repeat the above steps once. After discarding the supernatant, 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403) was added to Dynabeads, and 20 ⁇ l of BA was added.
  • BA buffer
  • the mixture was heated in a 55 ° C water bath for 30 minutes, and the centrifuge tube was placed in the corresponding sample well of the magnetic separator, allowed to stand for two minutes, and the supernatant was taken to obtain the separated free DNA.
  • Positive control The same amount of DNA (free DNA using SEQ ID NO. 9, representing the length of free DNA of 166 bp) was dissolved in 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403), and 20 ⁇ l of BA was added.
  • the DNA captured by the present invention, the DNA captured by the Kaijie kit, and the positive control were used for quantitative analysis of DNA using the Agilent 2100 High Sensitivity DNA Analysis Kit (Cat. 5067-4626), and the amount of DNA captured and grasped was obtained.
  • the percentage results are summarized in Table 5.
  • the extraction efficiency is the ratio of the amount of DNA captured by the present invention or the Kaijie kit to the amount of positive control DNA in Table 5 (i.e., the percentage of DNA grabbed).
  • the free DNA extraction efficiency of the invention is close to 80%, and The Circulating Nucleic Acid Kit is only 51%, so the extraction efficiency of extracting free DNA in plasma is increased by 54.7%.
  • Example 8 Binding of dCas9 protein conjugated to AVI-Tag to solid phase beads, binding exogenous free DNA in plasma and separating it
  • First step Purification of the his-S.P.dCas9-AVI protein, referring to Example 3, replacing the FLAG sequence with the AVI-Tag sequence (DNA sequence: ggcctgaacgatattttttgaagcgcagaaaattgaatggcatgaa).
  • Step 2 The AVI-Tag-dCas9 protein was biotinylated using the AViDITY BirA-500kit (Cat. Bir A500) according to the kit instructions.
  • the reaction was carried out by adding the corresponding reactants to a 1.5 ml centrifuge tube as follows:
  • BirA enzyme 0.8 ⁇ l
  • the reaction solution was then allowed to react at room temperature for 1 hour or overnight.
  • Step 3 The biotinylated protein is linked to Streptavidin gel beads.
  • the fourth step after the reaction is completed, centrifuge at 5000 RPM for 30 seconds, discard the supernatant, add 150 ⁇ l of the buffer (BA) in Example 4, mix well, centrifuge at 5000 RPM for 30 seconds, discard the supernatant, and repeat the above steps. once. After discarding the supernatant, 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403) was added to the gel beads, and 20 ⁇ l of BA was added. After mixing uniformly, the mixture was heated in a 55 ° C water bath for 30 minutes, centrifuged at 5000 RPM for 30 seconds, and the supernatant was taken to obtain isolated free DNA.
  • BA buffer
  • RT403 proteinase K
  • Positive control The same amount of DNA (free DNA using SEQ ID NO. 9, representing the length of free DNA of 166 bp) was dissolved in 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403), and 20 ⁇ l of BA was added.
  • the DNA captured by the present invention, the DNA captured by the Kaijie kit, and the positive control were used for quantitative analysis of DNA using the Agilent 2100 High Sensitivity DNA Analysis Kit (Cat. 5067-4626), and the amount of DNA captured and grasped was obtained.
  • the percentage results are summarized in Table 6.
  • the extraction efficiency is the ratio of the amount of DNA captured by the present invention or the Kaijie kit to the amount of positive control DNA in Table 6 (i.e., the percentage of DNA grabbed).
  • the free DNA extraction efficiency of the invention is close to 78%, and The Circulating Nucleic Acid Kit is only 51%, so the present invention increases the extraction efficiency of extracting free DNA in plasma by 53%.
  • Example 9 The binding of His-Tag-containing Cas9 protein to solid phase beads, binding of exogenous free DNA to plasma and separation thereof
  • Step 1 Cas9 protein with His-Tag (IDT, SpCas9Nuclease V3, Cat.No.1081058) with His-Tag separating beads (Dynabeads TM His-Tag Isolation and Pulldown, Cat.No.10103D) connection.
  • IDT IDT
  • SpCas9Nuclease V3, Cat.No.1081058 His-Tag separating beads
  • His-Tag separating beads Dynabeads TM His-Tag Isolation and Pulldown, Cat.No.10103D
  • Step 2 After the reaction, the centrifuge tube containing plasma and Dynabeads was placed in the corresponding sample well of the magnetic separator, allowed to stand for two minutes, the supernatant was discarded, and 150 ⁇ l of the buffer (BA) in Example 4 was added to centrifuge. Place the tube in the sample well corresponding to the magnetic separator, let stand for two minutes, discard the supernatant, and repeat the above steps once. After discarding the supernatant, 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403) was added to Dynabeads, and 20 ⁇ l of BA was added.
  • BA buffer
  • the mixture was heated in a 55 ° C water bath for 30 minutes, and the centrifuge tube was placed in the corresponding sample well of the magnetic separator, allowed to stand for two minutes, and the supernatant was taken to obtain the separated free DNA.
  • Positive control The same amount of DNA (free DNA using SEQ ID NO. 9, representing the length of free DNA of 166 bp) was dissolved in 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403), and 20 ⁇ l of BA was added.
  • the DNA captured by the present invention, the DNA captured by the Kaijie kit, and the positive control were used for quantitative analysis of DNA using the Agilent 2100 High Sensitivity DNA Analysis Kit (Cat. 5067-4626), and the amount of DNA captured and grasped was obtained.
  • the percentage results are summarized in Table 7.
  • the extraction efficiency is the ratio of the amount of DNA captured by the present invention or the Kaijie kit to the amount of positive control DNA in Table 7 (i.e., the percentage of DNA grabbed).
  • the free DNA extraction efficiency of the invention exceeds 85%, and The Circulating Nucleic Acid Kit is only 55%, so the extraction efficiency of extracting free DNA in plasma is increased by 54.5%.
  • Example 10 The dCas9 protein conjugated with AVI-Tag binds to solid phase beads and binds to endogenous free DNA in plasma and separates it.
  • the first step and the second step were the same as the first step and the second step in Example 8, to obtain a biotinylated dCas9 protein.
  • Step 3 The biotinylated protein is linked to Streptavidin gel beads.
  • the fourth step after the reaction is completed, centrifuge at 5000 RPM for 30 seconds, discard the supernatant, add 150 ⁇ l of the buffer (BA) in Example 4, mix well, centrifuge at 5000 RPM for 30 seconds, discard the supernatant, and repeat the above steps. once. After discarding the supernatant, 30 ⁇ l of proteinase K (Tiangen Biochemistry, RT403) was added to the gel beads, and 20 ⁇ l of BA was added. After mixing uniformly, the mixture was heated in a 55 ° C water bath for 30 minutes, centrifuged at 5000 RPM for 30 seconds, and the supernatant was taken to obtain isolated free DNA.
  • BA buffer
  • RT403 proteinase K
  • the DNA captured by the present invention was subjected to quantitative analysis of DNA using the Agilent 2100 High Sensitivity DNA Analysis Kit (Cat. 5067-4626) together with the DNA captured by the Kaijie kit, and the obtained amount of grasped DNA was summarized in Table 8. in.
  • the free DNA extraction amount of the invention is close to 28.49 pg/ ⁇ l, and The Circulating Nucleic Acid Kit is only 24.68 pg/ ⁇ l, so the extraction efficiency of extracting endogenous free DNA in plasma is increased by 15.4%.

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Abstract

本发明涉及一种从溶液中分离DNA的方法，尤其涉及一种采用Cas蛋白从溶液中分离DNA的方法和试剂盒。本发明能有效提高提取游离DNA的效率。

Description

一种Cas蛋白体系分离DNA的方法 技术领域
本发明涉及一种分离DNA的方法，尤其涉及一种采用Cas蛋白从溶液中分离DNA的方法。
背景技术
通常情况下，绝大多数脱氧核糖核酸(DNA)位于细胞中，但不管是在微生物、植物还是动物的细胞外，也常常存在着DNA的片段，这类核酸被统称为游离DNA。Mandel和Metasis在1948年首次报道了在外周血中发现游离DNA(Mandel P.C R Hebd Seances Acad Sci(Paris),1948,142(3):241-3)。经过半个世纪的研究，科学家发现不论是健康人群还是患病人群，都有小部分DNA游离于细胞外。这类DNA通常存在于血液、血浆、血清和其他体液中。在各个样品中发现的细胞外DNA由于被核酸酶所保护(例如它们以蛋白脂质复合物的形式被分泌出来，常常与蛋白质结合，或被包含在囊泡中)，因此并不容易被降解。在患有疾病，如恶性肿瘤和传染病的人群中，这类细胞外DNA的含量常常比正常人要高很多，因此在疾病筛查、诊断、预后、病程进展监测、甚至是治疗靶标的识别等方面都有很大的应用前景。此外，母体血液中常伴随有相当含量的胎儿DNA，这类DNA可以用于多种检测，例如性别鉴定，染色体异常的测试和评估，同时还能监测妊娠相关的并发症。也正是由于其较强的临床诊断相关性，游离DNA在无创诊断和预后方面有着巨大的应用潜力，可用于例如无创产前基因检测、肿瘤检测、移植医学等许多其他疾病。
目前，许多从各种生物液体中提取并分析单链或双链的游离DNA的方法已经被报道，其中包括阴离子交换法(WO2016198571A1)，二氧化硅珠提取法(WO2015120445A1)，曲拉通/加热/苯酚法(Xue et al.Clinica Chimica Acta 2009.404:100-104)等。这些方法通常会使用分解离液剂将游离DNA从蛋白质的包裹中释放出来，然后通过物理吸附或析出的方法将游离DNA分离出来。然而，使用这些方法提取DNA的产率非常低，很难从大 样本中提取少量DNA。例如，目前市售的用于游离核酸提取的
Figure PCTCN2018125973-appb-000001
血清/血浆核酸纯化试剂盒(Circulating Nucleic Acid kit，QIAGEN(凯杰)公司)，对游离DNA的提取效率只有不到50％。
成簇规律间隔短回文重复序列(CRISPR)和CRISPR结合蛋白(Cas蛋白)组成了一套强大的核酸酶系统，能够对真核细胞中几乎所有与原型间隔子邻近基序(protospacer-adjacent motif,PAM)相邻的基因组序列进行切割(Cong et al.Science 2013.339:819-823)。到目前为止，所有和CRISPR/Cas系统相关的应用除了需要Cas蛋白之外，还包含了RNA组分。所述RNA组分是由一条CRISPR RNA(crRNA)和一条反式激活crRNA(tracrRNA)组成的双链指导RNA结构，指导Cas蛋白切割与crRNA序列互补的DNA位点(Jinek et al.Science 2012.337:816-821)。为了进一步简化CRISPR/Cas9系统，研究人员利用工程学方法将双链指导RNA的两个组分(crRNA和tracrRNA)的部分连接成嵌合型单链指导RNA(sgRNA)。这体现了CRISPR/Cas9系统强大的基因识别和基因编辑能力，但是，Cas蛋白本身的性质并没有被深入的研究。到目前为止，还没有研究人员发现单独使用作为核酸酶的Cas蛋白及其变体在识别并结合DNA上的潜力。
目前，市场上存在对高效提取游离DNA方法的强烈需求，以便从各种生物液体的大样本中高效提取游离DNA，用于后续分析检测。
本发明的简述
为了提高对溶液中DNA的提取效率，本发明提供了如下技术方案：
(1)一种采用Cas蛋白从溶液中分离DNA的方法。
(2)根据技术方案(1)所述的方法，包括步骤a)DNA溶液与Cas蛋白体系混合；c)从Cas蛋白体系与DNA结合的复合物中分离得到DNA
(3)根据技术方案(1)所述的方法，包括步骤a)在DNA的溶液中加入Cas蛋白体系；b)富集步骤a)中的Cas蛋白体系与DNA结合的复合物；c)从步骤b)富集的复合物中分离得到DNA。
(4)根据技术方案(2)所述的方法，其中所述的Cas蛋白体系为偶联了平板状、膜状或柱状的固体支持物的Cas蛋白。
(5)根据技术方案(3)所述的方法，其中所述的Cas蛋白体系为偶联了珠形固体支持物的Cas蛋白。
(6)根据技术方案(3)所述的方法，其中所述的Cas蛋白体系为Cas蛋白与至少一种连接序列形成的Cas融合蛋白。
(7)根据技术方案(6)所述的方法，其中步骤b)在溶液中加入偶联了亲和分子的固体支持物，再通过离心富集或磁性富集。
(8)根据技术方案(6)所述的方法，其中在步骤a)中加入偶联了亲和分子的固体支持物，孵育后，再通过步骤b)离心富集或磁性富集。
(9)根据技术方案(1)-(8)任意一项所述的方法，其中所述的DNA是双链DNA。
(10)根据技术方案(4)或(5)所述的方法，其中所述的Cas蛋白体系为Cas蛋白通过化学共价键偶联到固体支持物。
(11)根据技术方案(6)所述的方法，其中所述的Cas蛋白体系为Cas融合蛋白与固体支持物上的亲和分子结合而形成的。
(12)根据技术方案(4)-(11)任一项所述的方法，其中所述的固体支持物是能与氨基酸残基形成化学键的固体物质。
(13)根据技术方案(10)所述的方法，其中所述的固体支持物选自至少一种凝胶材料、磁性材料、纤维素材料、硅胶材料、玻璃材料和人造高分子聚合物制得的固体物质。
(14)根据技术方案(5)-(13)任一项所述的方法，其中所述的固体支持物为磁珠、凝胶珠和/或硅胶珠。
(15)根据技术方案(6)-(14)任一项所述的方法，其中所述的连接序列和亲和分子选自下组配体和受体组合中的一种，酶与底物、抗原与抗体、生物素与亲和素。
(16)根据技术方案(15)所述的方法，其中所述的酶与底物选自谷胱甘肽转移酶与谷胱甘肽。
(17)根据技术方案(15)所述的方法，其中所述的抗原-抗体选自下组中的一种：A蛋白或其保留了结合Fc区域功能性的片段与免疫球蛋白或其Fc或Fab片段、G蛋白或其保留了结合Fc区域功能性的片段与免疫球蛋白或其Fc或Fab片段、组氨酸标签与抗组胺酸标签、多组氨酸标签与抗多组氨酸标签和FLAG标签与抗FLAG标签。
(18)根据技术方案(15)所述的方法，其中所述的生物素与亲和素选自下组中的一种：生物素与亲和素或链酶亲和素、连接了生物素的生物素受体蛋白与亲和素或链酶亲和素、Strep-tag与亲和素或链酶亲和素。
(19)根据前述技术方案(2)-(18)任意一项所述的方法，其中所述的步骤a)的反应时间为5分钟到6小时，优选反应时间为15分钟到1小时。
(20)根据前述技术方案(2)-(18)任意一项所述的方法，其中所述的步骤a)的反应温度为20-37℃，优选反应温度为30-37℃。
(21)根据前述技术方案(7)、(12)-(20)任意一项所述的方法，其中所述的步骤b)，在溶液中加入偶联了亲和分子的固体支持物的反应时间为30分钟至过夜，优选反应时间为1-2小时。
(22)根据技术方案(21)所述的方法，其中所述的步骤b)的反应温度为4℃到37℃，优选反应温度为20-30℃。
(23)根据前述技术方案(3)-(22)任意一项所述的方法，其中所述b)的富集为离心富集或磁性富集。
(24)根据前述技术方案(2)-(23)任意一项所述的方法，其中所述步骤c)通过洗脱分离DNA。
(25)根据前述技术方案(2)-(24)任意一项所述的方法，其中所述的Cas是具有DNA结合功能的Cas蛋白。
(26)根据技术方案(25)所述的方法，其中所述的Cas蛋白是天然的Cas蛋白、突变后失去了gRNA结合能力的Cas蛋白，或者突变后核酸酶失活的Cas蛋白。
(27)根据技术方案(25)或(26)所述的方法，其中所述的Cas蛋白是Cas9蛋白。
(28)根据技术方案(27)所述的方法，其中所述的Cas9蛋白是dCas9蛋白。
(29)一种从溶液中分离DNA的试剂盒，包含组分a)Cas蛋白体系，和组分b)DNA洗脱液。
(30)根据技术方案(28)所述的试剂盒，其中组分a)Cas蛋白体系是偶联了固体支持物的Cas蛋白溶液体系，或是含有Cas融合蛋白和偶联了亲和分子的固体支持物的溶液体系。
(31)根据技术方案(29)所述的试剂盒，其中组分a)Cas蛋白体系由两个独立的体系组成，i)Cas蛋白与至少一种连接序列形成的Cas融合蛋白，和ii)偶联了亲和分子的固体支持物。
(32)根据技术方案(29)所述的试剂盒，其中组分b)DNA洗脱液可以是以下液体：0.5-10mg/ml蛋白酶K溶液、1-5M浓度的NaCl或者KCl盐溶液、具有0-0.3M Tris和0-0.5M NaCl的pH 11-12.5的碱洗脱液。
(33)根据技术方案(30)或(31)所述的试剂盒，其中的固体支持物为平板状、膜状或柱状的固体支持物。
(34)根据技术方案(33)所述的试剂盒，其中的固体支持物选自凝胶材料、磁性材料、纤维素材料、硅胶材料、玻璃材料和人造高分子聚合物制得的固体物质中的至少一种。
(35)根据技术方案(34)所述的试剂盒，其中的固体支持物为磁珠、凝胶珠和/或硅胶珠。
(36)根据技术方案(31)-(35)所述的试剂盒，其中所述的连接序列和亲和分子选自下组配体和受体组合中的一种：酶与底物、抗原与抗体、生物素与亲和素。
(37)根据技术方案(36)所述的试剂盒，其中所述的与底物选自谷胱甘肽转移酶与谷胱甘肽。
(38)根据技术方案(37)所述的试剂盒，其中所述的抗原-抗体选自下组中的一种：A蛋白或其保留了结合Fc区域功能性的片段与免疫球蛋白或其Fc或Fab片段、G蛋白或其保留了结合Fc区域功能性的片段与免疫球蛋白或其Fc或Fab片段、组氨酸标签与抗组胺酸标签、多组氨酸标签与抗多组氨酸标签和FLAG标签与抗FLAG标签。
(39)根据技术方案(36)所述的试剂盒，其中所述的生物素与亲和素选自下组中的一种：生物素与亲和素或链酶亲和素、连接了生物素的生物素受体蛋白与亲和素或链酶亲和素、Strep-tag与亲和素或链酶亲和素。
(40)根据技术方案(29)-(39)所述的试剂盒，其中所述的Cas是具有DNA结合功能的Cas蛋白。
(41)根据技术方案(40)所述的试剂盒，其中所述的Cas蛋白是天然的Cas蛋白、突变后失去了gRNA结合能力的Cas蛋白，或者突变后核酸酶失活的Cas蛋白。
(42)根据技术方案(41)所述的试剂盒，其中所述的Cas蛋白是Cas9蛋白。
(43)根据技术方案(42)所述的试剂盒，其中所述的Cas9蛋白是dCas9蛋白(突变后核酸酶失活的Cas9蛋白)。
在同等含量的DNA样本条件下，本发明中的提取方法省去了繁琐的样本处理过程，减少了样本的使用量要求，同时大幅提高了提取游离DNA的效率。
附图说明
图1：包含his-S.P.dCas9-FLAG基因片段的表达载体pSMART-his-S.P.dCas9-FLAG
图2：486bp的游离DNA(SEQ ID NO.8)与dCas蛋白的结合电泳条带。
泳道1是游离DNA自身的条带
泳道2是dCas9和游离DNA(SEQ ID NO.8)结合之后的情况
本发明的详述
Cas蛋白
成簇规律间隔短回文重复序列(CRISPR)和CRISPR结合蛋白(Cas蛋白)组成了一套强大的核酸酶系统。其中CAS蛋白可分为四个不同的功能模块：目标识别模块(间隔获取)；表达模块(crRNA加工和靶标结合)；干扰模块(靶标切割)；和辅助模块(监管和其他CRISPR相关功能)。近年来，核心Cas蛋白(Cas1-Cas10)已经积累了大量的结构和功能信息，这使得它们可以被分类到这些模块中。CRISPR相关内切酶Cas蛋白可以通过单链指导RNA(sgRNA)靶向特定的基因组序列。以化脓链球菌(Streptococcus pyogenes)Cas9蛋白(spCas9)蛋白为例，该蛋白包含有目标识别模块和核酸酶模块(干扰模块)组成的双叶结构，其在界面处的带正电的凹槽中容纳sgRNA-DNA异源双链体。而目标识别区对于结合sgRNA和DNA是必需的，核酸酶区包含HNH和RuvC核酸酶结构域，它们分别适合于切割靶DNA的互补链和非互补链。核酸酶区还含有负责与原型间隔子相邻基序(PAM)相互作用的羧基末端结构域。但是，到目前为止，还没有任何研究组发表关于Cas蛋白直接结合DNA的结构区域，或者Cas-DNA复合物的晶体结构。
因此在本发明中，在没有RNA介导的情况下，Cas蛋白本身就是一种核酸结合系统。通常情况下，Cas蛋白在没有RNA介导时便能够识别并结合几乎所有的DNA序列，同时并不对所结合的DNA序列进行剪切。
本发明所述的Cas蛋白是具有DNA结合功能的Cas蛋白，例如天然的Cas蛋白，突变的Cas蛋白，突变后失去了gRNA结合能力的Cas蛋白，突变后核酸酶失活的Cas蛋白(dead Cas，dCas)。
Cas蛋白的非限制性实例包括：Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8，Cas9(也称为Csn1或Csx12)、Cas10、Cas12、Cas13、Cas14、Csy1、Csy2、Csy3、Cse1、Cse2、Csc1、Csc2、Csa5、Csn2、Csm2、Csm3、Csm4、Csm5、Csm6、Cmr1、Cmr3、Cmr4、Cmr5、Cmr6、Csb1、Csb2、Csb3、Csx17、Csx14、Csx10、Csx16、CsaX、Csx3、Csx1、Csx15、Csf1、Csf2、Csf3、Csf4其在不同物种中的同源蛋白、核酸内切酶失活的突变蛋白、或其修饰形式。
在一个具体实施方式中，本发明的Cas蛋白是Cas9蛋白，更优选突变后核酸酶失活的Cas9蛋白(dead Cas9，dCas9)
Cas9，也称为Csn1或Csx12，是既参与crRNA生物合成又参与摧毁入侵DNA的巨型蛋白质。已经在不同的细菌物种如嗜热链球菌(S.thermophiles)、无害利斯特氏菌(Listeria innocua)(Gasiunas，Barrangou et al.2012；Jinek，Chylinski et al.2012)和化脓性链球菌(S.Pyogenes)(Deltcheva，Chylinski et al.2011)中描述了Cas9。本发明的Cas9包括但不限于以下类型：化脓链球菌(Streptococcus pyogenes)Cas9蛋白，其氨基酸序列见SwissProt数据库登录号Q99ZW2；脑膜炎奈瑟氏菌(Neisseria meningitides)Cas9蛋白，其氨基酸序列见UniProt数据库编号A1IQ68；嗜热链球菌(Streptococcus thermophilus)Cas9蛋白，其氨基酸序列见UniProt数据库编号Q03LF7；金黃色葡萄球菌(Staphylococcus aureus)Cas9蛋白，其氨基酸序列见UniProt数据库编号J7RUA5。
作用于DNA双链的Cas蛋白一般具有两个内切酶活性位点，如果有且只有一个位点发生突变或缺失造成该位点酶活失效，则可以得到切割DNA单链的Nickase，例如Cas9-nickase，因其高保真率以及切割DNA单链的特性而被广泛应用。
另外通过突变筛选出Cas蛋白变体存在一个或多个点突变的情况被证明可以提高其特异性，降低基因组编辑的脱靶率，或使兼容更多样的PAM序列，例如eSpCas9, SpCas9-HF，HeFSpCas9和HIFI-SpCas9，xCas9(Christopher A.V et al.Nature Medicine 2018(24),pp1216–1224；Johnny H.H et al.Nature 2018 556(7699),pp57-63)等。
鉴于Cas蛋白包含多个功能模块，其蛋白序列较长，为了利于包装转运以及功能的控制，其蛋白的序列也会通过蛋白质工程研究被截短从而形成Cas蛋白系统，例如Split-SpCas9系统包含截短蛋白形成的复合体(Jana M et al.Plant Biotechnology Journal 2017 15,pp.917–926)。
在一些实施例中，该Cas蛋白是含有上述蛋白或其突变体的融合蛋白或蛋白复合物，包括但不限定于在Cas上连接其他氨基酸序列的变体。在以上Cas蛋白或其变体基础上融合其他的功能性蛋白，可以增加Cas蛋白功能的特异性和有效性，也可以对基因组产生切割之外的效应。例如在Cas或dCas上融合FokI，这样可以增加Cas9蛋白对基因组切割的特异性，因为FokI只有二聚化才会有切割活性，这样就要求有一对识别区域才能完成切割进而减少了脱靶率。又例如在截去部分功能结构域但保存其DNA结合能力的Cas蛋白上连接FokI(Ma et al.ACS Synth.Biol.,2018,7(4),pp 978–985)。又例如在Cas-nickase或dCas上融合碱基的修饰酶(如脱氨酶，胞嘧啶脱氨酶，腺嘌呤脱氨酶)，这样可以高效的对基因组靶点区域进行定向的碱基修改(Komor et al.Sci.Adv.2017.3:eaao4774)。又例如对dCas9融合一些可以调节基因表达的蛋白结构域，可以有效对靶点基因进行表达调节，例如融合VP64，VPR等转录激活因子的dCas9可以在gRNA的引导下结合到靶向基因附近而激活表达；相反的，如果融合了转录抑制因子(如SRDX)的dCas9会对靶基因产生下调作用。dSpCas9-Tet1和dSpCas9-Dnmt3a可以用于修改表观遗传状态，调节内源基因启动子的甲基化状态来调节蛋白表达。
具有DNA结合功能的Cas蛋白
具有DNA结合功能的Cas蛋白可以是天然的Cas蛋白，或者在Cas蛋白的DNA结合功能区以外做的突变，或者在DNA结合功能区进行突变后获得的Cas蛋白。
检测Cas蛋白是否具有DNA结合功能，可以采用凝胶电泳法对比结合蛋白的DNA和未结合蛋白的DNA的胶图条带位置来判别(检测方法参见实施例1)。
核酸酶失活的突变Cas蛋白(dCas蛋白)
核酸酶失活的突变Cas蛋白(dCas蛋白)是Cas蛋白经突变而获得的变体，其核酸内切酶活性失活或基本失活，使Cas蛋白失去或者基本失去核酸内切酶活性，进而无法切割靶序列。前述列举的Cas蛋白非限制性实例都可通过核酸内切酶失活突变改造为dCas蛋白，所述突变包括一个或多个氨基酸残基的插入、缺失或取代等。
例如，某些Cas9突变可以使Cas9蛋白失去或者基本失去核酸内切酶活性，进而无法切割靶序列。对于某一物种的Cas9，例如spCas9而言，降低或消除核酸内切酶活性的示例性突变包括以下位置中的一个或多个突变：D10，G12，G17，E762，H840，N854，N863，H982，H983，A984，D986，或A987。文献证明导向RNA(gRNA)介导的核酸内切酶失活突变Cas9(称为dCas9)可导致大肠杆菌特异性内源基因、以及人细胞中EGFP报告基因的表达抑制(Qi et al.Cell 2013.152:1173-1183)。这项研究证明了使用gRNA介导的dCas9技术可准确的识别并结合相应的基因组。
dCas蛋白可以是非天然存在于自然界中，并且是由蛋白质工程或通过随机诱变获得的变体，其核酸内切酶活性失活或基本失活。例如，可以通过突变，即化脓性链球菌Cas9核酸内切酶的氨基酸序列中至少一个残基的缺失或插入或取代获得相应dCas9蛋白。
在一些实施例中，dCas9蛋白可以为不同物种的Cas9经核酸内切酶活性失活或基本失活突变而获得的变体，包括但不限于，化脓链球菌(Streptococcus pyogenes)Cas9蛋白、脑膜炎奈瑟氏菌(Neisseria meningitides)Cas9蛋白、嗜热链球菌(Streptococcus thermophilus)Cas9蛋白、金黃色葡萄球菌(Staphylococcus aureus)Cas9蛋白、肺炎链球菌Cas9蛋白等。
连接序列和亲和分子
本发明所述的连接序列是指直接或间接连接于Cas蛋白的C端或者N端的分子，其最终与Cas蛋白形成融合蛋白。
本发明所述的亲和分子是偶联在固相物质上的，能与前述Cas蛋白上的连接序列形成特异性结合的分子。
本发明的连接序列和亲和分子互为受体和配体关系。任何蛋白质连接序列都可以应用在本发明的实践中，只要它可与亲和分子特异性结合。连接序列和亲和分子选自下列配体受体组合中的一种：酶与底物、抗原-抗体、生物素与亲和素。其中所述的酶与底物选自下组中的一种：谷胱甘肽转移酶与谷胱甘肽；所述的抗原-抗体选自下组中的一种：A蛋 白或其保留了结合Fc区域功能性的片段与免疫球蛋白或其Fc或Fab片段、G蛋白或其保留了结合Fc区域功能性的片段与免疫球蛋白或其Fc或Fab片段、组氨酸标签与抗组胺酸标签、多组氨酸标签与抗多组氨酸标签、FLAG标签(氨基酸序列：ATLAAAAL)与抗FLAG标签；所述的生物素与亲和素选自下组中的一种：生物素(biotin)与亲和素(Avidin,UniProt数据库编号P02701)或链酶亲和素(Streptavidin,UniProt数据库编号P22629)、连接了生物素的生物素受体蛋白(如AviTag，氨基酸序列：GLNDIFEAQKIEWHE)与亲和素或链酶亲和素、Strep-tag(氨基酸序列：WSHPQFEK)与亲和素或链酶亲和素。
在本发明中，连接序列和亲和分子通过亲和作用特异性结合，并且可以互换。比如，如果连接序列是受体，则亲和分子为与该受体特异结合的配体；连接序列也可是所述配体，那么，亲和分子为与该配体特异结合的受体。又比如，如果连接序列为链酶亲和素，则亲和分子为生物素；连接序列也可以为生物素，那么亲和分子为链酶亲和素。
固相支持物
本发明所述的固相物质是能与氨基酸残基形成化学键的固体物质，因而可以与亲和分子或者Cas蛋白通过化学键偶联。
合适的固相支持物从材料区分可以是凝胶材料、磁性材料、纤维素材料、硅胶材料、玻璃材料，或人造高分子聚合物制得的固体物质。凝胶材料可以是水凝胶、有机凝胶、干凝胶、或纳米复合水凝胶；磁性材料可以是由高分子材料包裹的金属氧化物(如铁钴镍的氧化物)；纤维素材料可以是纤维素、醋酸纤维素、硝酸纤维素；硅胶材料可以是二氧化硅支持物或者有机硅胶支持物；人造高分子聚合物可以是、尼龙、聚醋、聚醚砜、聚烯烃、聚偏1，1-二氟乙烯和它们的组合。固相支持物从形式上区分可以是珠形、化学膜类柱、玻璃平板、6孔/24孔/48孔/96孔/384孔板，PCR反应管等。
结合了DNA的Cas蛋白与固体支持物，例如化学膜类柱、玻璃平板、6孔/24孔/48孔/96孔/384孔板、PCR反应管结合后，通过洗脱液将DNA洗脱下来。
结合了DNA的Cas蛋白与珠形的固体支持物结合后，经过富集，例如离心富集或磁性富集后，通过洗脱液将DNA洗脱下来。
本发明所述的固体支持物优选是珠形的，例如磁珠、凝胶珠和/或硅胶珠。可以使用的凝胶珠非限制性实例包括：Anti-6X His
Figure PCTCN2018125973-appb-000002
antibody(琼脂糖凝胶)(Abcam)，Anti- 6xHis
Figure PCTCN2018125973-appb-000003
Acceptor beads(PerkinElmer)，Dynabeads TM His-Tag Isolation and Pulldown(ThermoFisher)，Anti-His-tag mAb-Magnetic Beads(MBL Life science)，His Tag Antibody Plate(GenScript)。
偶联
可以采用常规的方法将亲和分子或Cas蛋白偶联到固体支持物上。将A蛋白和G蛋白连接到固体支持物上，可以参见，例如Hermanson et al.1992，Immobilized Affinity Ligand Techniques，Academic Press。典型地，固体支持物是用反应官能团(“活化基团”)如环氧化物(表氯醇)、氰(溴化氰CNBr)，N，N-二琥珀酰亚胺基碳酸酯(DSC)、醛或活化的羧酸(如N-羟基丁二酰亚胺(NHS)酯、羰基二咪唑(CDI)活化酯)来活化的。这些活化基团能直接连接到固体支持物上，如CNBr，或者它们也可以是“接头”或间隔分子的一部分，典型地是碳、氧和氮原子的直链，如在接头丁二醇二缩水甘油醚(一种常用的环氧化物偶联剂)中发现的碳和氧的十元链。在偶联条件下再将活化的固体支持物与Cas蛋白质或亲和分子相平衡。偶联反应完成，彻底洗涤介质。
Cas融合蛋白
本发明的Cas融合蛋白是将连接序列添加到Cas蛋白的N端或C端或其蛋白序列中间任意部分而形成的融合蛋白。本发明的融合Cas蛋白可以通过基因工程的方法，使Cas蛋白和连接序列一起表达。
Cas蛋白体系
本发明所述的Cas蛋白体系是一种包含Cas蛋白的复合物，用于结合溶液中的DNA。
在一个具体的实施方式中，Cas蛋白体系是Cas蛋白通过化学共价键偶联到固体支持物上而形成的复合物。
在一个具体的实施方式中，Cas蛋白体系是Cas蛋白与一个或多个连接序列形成的Cas融合蛋白。
在另一个具体的实施方式中，Cas蛋白体系是Cas融合蛋白与固相物质上偶联的亲和分子结合后形成的复合物。
Cas融合蛋白和偶联亲和分子的固相物质的结合
Cas蛋白与连接序列一同组成Cas融合蛋白。连接序列可直接或间接位于Cas蛋白的N端或C端。以固相物质为凝胶珠为例，在一种优选的实施方式中，使用偶联亲和分子的凝胶珠结合Cas融合蛋白时，对于每10μl的凝胶珠，在加入凝胶珠之前，反应液中Cas融合蛋白的初始浓度为0.001-20μg/μl，优选为0.01-10μg/μl的Cas9融合蛋白。
Cas融合蛋白与固相物质的结合在4℃到37℃进行，优选4℃或室温(20-25℃)。采用的结合反应液可以为去离子水，1倍磷酸盐缓冲生理盐水(Phosphate buffered saline：PBS)，Tris缓冲液(50mM Tris-HCl，150mM NaCl，pH7.4)，或者其他盐离子浓度低于150mM、不含油去垢剂和变性剂的pH为约中性的缓冲液。反应时间为30分钟到过夜，优选过夜。
Cas蛋白也可以不经连接蛋白而直接与偶联亲和分子的固相物质结合。
Cas蛋白和游离DNA的结合
本发明的Cas蛋白可以结合任何游离状态的DNA。该DNA可以是双链或者单链DNA。与Cas蛋白结合的游离DNA可通过固相物质结合Cas蛋白的方式将其分离出液相。
与本发明的Cas蛋白结合的DNA可以为任何游离DNA。例如，以基因组为模板经PCR得到的任意序列DNA片段、序列为人工设计的DNA片段、细胞裂解后游离的DNA、细胞裂解后得到的基因组、人体或动物体液中游离的DNA。在一些实施方式中，DNA是以基因组为模板经PCR得到的DNA片段、序列为人工设计的DNA片段，或人体或动物体液中游离的DNA。
作为优选的实施方式，Cas蛋白或Cas融合蛋白结合游离DNA时，DNA结合反应液中蛋白浓度为0.001-20μg/μl。优选0.01-10μg/μl的Cas蛋白或其变体或Cas融合蛋白。在一种优选的实施方式中，反应在室温(20-25℃)到37℃进行。采用的DNA结合反应液可以为去离子水，1倍磷酸盐缓冲生理盐水(Phosphate buffered saline：PBS)，Tris缓冲液(50mM Tris-HCl，150mM NaCl，pH7.4)，或者其他盐离子浓度低于150mM、不含油去垢剂和变性剂的pH为中性的缓冲液。优选为50mM KCl，10mM EDTA，30mM Tris-HCl，0.2％Triton X-100，12％甘油(glycerol)。反应时间为5分钟到6小时，在一 种实施方式中，优选时间为5分钟到2小时。在另一种实施方式中，优选时间为15分钟到1小时。
将游离DNA从溶液中分离出来的方法
本发明的Cas蛋白分离游离DNA的方法能有三种使用方式：
方法一，Cas蛋白或Cas融合蛋白先在DNA结合反应液中与游离DNA结合，之后，再在蛋白结合反应液中通过偶联亲和分子的固相物结合Cas蛋白或Cas融合蛋白。首先，在前面所述DNA结合反应条件下将Cas蛋白或Cas融合蛋白与游离DNA在DNA结合反应液孵育结合，优选的时间为5分钟到6小时，在另一种实施方式中，优选时间为15分钟到1小时。反应温度为室温(20-25℃)到37度；DNA结合反应结束后，直接在溶液中加入偶联亲和分子的固相物质，如前面所述蛋白质结合反应条件下，使Cas蛋白或Cas融合蛋白与偶联亲和分子的固相物质结合，优选反应时间为30分钟到过夜，反应温度为4℃到37℃；最后通过离心分离固相物质，弃上清；在清洗固相物质数次之后，将Cas蛋白或其变体或Cas融合蛋白以及结合的DNA从固相物质上洗脱下来，得到洗脱溶液，可以使用的洗脱液为含0.5-10mg/ml蛋白酶K溶液、1-5M浓度的NaCl或者KCl盐溶液、碱洗脱液(0-0.3M Tris，0-0.5M NaCl，pH 11-12.5)、酸洗脱液(0-0.3M甘氨酸HCl，pH 2.5-3.5)、竞争洗脱液(DYKDDDK或者FLAG氨基酸的TBS溶液，氨基酸浓度可以是100到500μg/ml)，或PAGE胶样本溶液(0.01M Tris-HCl，10％Glycerol，0.016％溴酚蓝)。
方法二，偶联亲和分子的固相物质首先结合Cas蛋白或其变体或Cas融合蛋白，再于DNA结合反应液中孵育结合游离DNA。使用的反应条件和溶液、洗脱条件和方法一一致。
方法三，将偶联亲和分子的固相物质、Cas9蛋白或其变体或Cas融合蛋白同时加入DNA结合反应液中，共同孵育后分离固相物质。所述方法中的溶液可以是上述Cas蛋白或其变体或Cas融合蛋白结合游离DNA使用的溶液，优选反应时间为30分钟到6小时，反应温度为室温(20-25℃)到37度。洗脱条件和方法一一致。
实施例
实施例仅为举例说明，不旨在对本发明造成任何方式上的限制。
实施例1：Cas蛋白的DNA结合活性
Cas蛋白的DNA结合活性可以采用凝胶电泳法，对比结合Cas蛋白的DNA和未结合Cas蛋白的DNA的胶图条带位置来判别。采用如下反应条件：
反应缓冲液(BA)：50mM三羟甲基氨基甲烷盐酸盐(Tris-HCl,pH 7.4)；150mM氯化钠(NaCl)
反应体系(20μl)：
dCas蛋白：2μg
DNA：0.25μg
无核酸酶双蒸水：定容到20μl
反应过程：
上述反应体系加入200μl PCR反应管中混合均匀后，室温或37℃反应15分钟到1小时。反应结束后，使用非变性胶，在BioRad电泳槽中，以80V电压，400mA电流运行80分钟。取出非变性胶后用DuRed(上海翊圣生物科技有限公司，10202ES76)将核酸染色，继而使用凝胶成像仪拍摄取得图片(见图2)。结合Cas蛋白的DNA的电泳条带明显滞后于未结合Cas蛋白的DNA的电泳条带。
实施例2：游离DNA的制备
本发明中的游离DNA可以是任意序列的DNA。在本实施例中，我们合成、构建了不同长度的11种DNA序列，如表1所示。
表1：游离DNA序列(5’-3’)
SEQ ID NO: 长度(bp)
1 100
2 100
3 100
4 200
5 200
6 200
7 957
8 486
9 166
10 609
11 2655
SEQ ID NO.1-6号DNA序列通过正反向引物(表2)直接PCR而合成，并未使用任何模板。使用这六个DNA的主要目的是想要检测原型间隔子相邻基序(PAM)区域是否会对Cas蛋白与DNA的结合产生影响。其中SEQ ID NO.1-3为100bp，4-6为200bp，其中1和4号序列上没有Cas9相应的PAM区域(NGG)，2和5号序列上各有一个PAM区域，而3和6号序列上各有3个PAM区域。详细结果会在实施例4中描述讨论。
SEQ ID NO.7-11号DNA序列则是通过分别设计相对应的正反向引物，以293T细胞(上海生科院，GNHu17)基因组作为模板PCR反应制成。
表2：不同游离DNA序列所对应的上下游引物，以及各自PCR反应的退火温度。
  上游引物 下游引物 退火温度(℃)
SEQ ID NO:1 SEQ ID NO：12 SEQ ID NO：13 55
SEQ ID NO:2 SEQ ID NO：14 SEQ ID NO：15 55
SEQ ID NO:3 SEQ ID NO：16 SEQ ID NO：17 55
SEQ ID NO:4 SEQ ID NO：18 SEQ ID NO：19 58
SEQ ID NO:5 SEQ ID NO：20 SEQ ID NO：21 58
SEQ ID NO:6 SEQ ID NO：22 SEQ ID NO：23 58
SEQ ID NO:7 SEQ ID NO：24 SEQ ID NO：25 68
SEQ ID NO:8 SEQ ID NO：26 SEQ ID NO：27 60
SEQ ID NO:9 SEQ ID NO：28 SEQ ID NO：29 60
SEQ ID NO:10 SEQ ID NO：30 SEQ ID NO：31 59
SEQ ID NO:11 SEQ ID NO：32 SEQ ID NO：33 66
SEQ ID NO.1-6号DNA的PCR反应使用上述的2条上下游引物与Q5热启动超保真2X Master Mix试剂盒(New England Biolabs,M0494S)进行，每条引物的终浓度为0.5 μM，加入10μl Q5 2X Master Mix，再加水至总体积为50μl，然后根据厂家的指南进行PCR。SEQ ID NO.7-11号DNA序列在上述反应体系的基础上，额外加入1μl细胞基因组提取液，最后定容至50μl。
实施例3：构建并纯化偶联了FLAG的dCas9蛋白
1蛋白表达
1.1表达载体构建：pSMART-his-S.P.dCas9-FLAG。以下为载体环状示意图，his-S.P.dCas9-FLAG基因片段(SEQ ID No:34)通过EcoRI和BamHI限制性核酸内切酶整合到pSMART载体上(见图1)；如果使用其他连接序列，则将载体中的FLAG(FLAG序列：GATTACAAGGATGACGATGACAAG)替换为相应的序列，如AVI-Tag，其他表达纯化蛋白的步骤相同：
1.2表达菌株选择：Rosetta2(NOVAGEN,17400)
1.3培养条件：摇瓶培养
仪器耗材：摇床，LB培养基(生工，A507002)
步骤：
从4度冰箱里拿出平板(Rosetta/BL21)活化，37度过夜；转接：500ml的LB接了菌液4ml，37度恒温培养4小时，OD600＝0.8，拿出放在室温，降摇床温度至18度，加入500ul的IPTG(异丙基-β-D-硫代吡喃半乳糖苷，天根RT108-01)，过夜诱导，诱导时间16小时。
2蛋白纯化
2.1菌体收获及裂解
仪器耗材：离心机，匀浆机，高速离心机，0.45μm滤膜(默克密理博，SLHV033)，裂解液
步骤：菌体经5000rpm，30分钟离心收集，加入裂解液进行重悬，经过匀浆机进行破碎。高速离心分离上清和沉淀。上清经过0.45μm的滤膜后准备进入下一步层析纯化。
2.2层析柱纯化流程：
仪器：AKTA-purify，层析柱，层析溶液
2.2.1组蛋白亲和层析
步骤：20ml组蛋白亲和层析柱直接用购买的介质(GE生命科学，17-5318-02)装柱，用0.5％的溶液B(20mM Tris-Hcl，PH＝8.0；250mM NaCl；1M咪唑)进行平衡。根据AKTA仪器说明操作得到纯化后蛋白溶液。
2.2.2阳离子交换层析
步骤：使用20ml阳离子交换层析柱(GE生命科学，17-0407-01)，用0.5％的溶液B(20mM HEPES-KOH PH＝7.5；1M KCL)对其进行平衡。根据AKTA仪器说明操作得到纯化后蛋白溶液。
3浓缩换液
仪器耗材：30kD浓缩管(默克密理博，UFC903096)，低温高速离心机
步骤：将蛋白溶液加入浓缩管中，4℃离心，5000rpm，40分钟，取出浓缩管，清除穿透液，加入15ml蛋白保存液(20mM Hepes，PH＝7.5；150mM KCl；1％蔗糖；30％甘油；1mM二硫苏糖醇(DTT))，4℃离心40分钟；重复上述步骤3次，得到最终his-S.P.dCas9-FLAG蛋白溶液，分装后放入-80℃保存。
实施例4：游离DNA和Cas蛋白结合
通常情况下，CRISPR/Cas系统会基于PAM区域(序列：NGG，N可以是A/T/C/G)以及RNA的指导对特定的DNA序列进行识别和切割。为了验证Cas蛋白及其变体与核酸的直接结合是否也依赖于PAM区域，我们制备了SEQ ID NO.1-6号DNA序列。其中1-3为100bp，4-6为200bp，其中1和4号序列上没有Cas9相应的PAM区域(NGG)，2和5号序列上各有一个PAM区域，而3和6号序列上各有3个PAM区域。反应所用的dCas9蛋白与实施5、6和7相同，即为实施例3中生产纯化的FLAG-dCas9蛋白。反应条件如下：
反应缓冲液(BA)：50mM三羟甲基氨基甲烷盐酸盐(Tris-HCl,pH 7.4)；150mM氯化钠(NaCl)
反应体系：
总体积：20μl
dCas9蛋白：2μg
DNA：0.25μg
无核酸酶双蒸水：定容到20μl
反应过程及结果：
上述反应体系加入200μl PCR反应管中混合均匀后，室温或37℃反应15分钟到1小时。反应结束后，使用非变性胶，在BioRad电泳槽中，以80V电压，400mA电流运行80分钟。取出非变性胶后用DuRed(上海翊圣生物科技有限公司，10202ES76)将核酸染色，继而使用凝胶成像仪拍摄取得图片(图2)。图2展示了500bp左右的游离DNA(SEQ ID NO.7)与dCas蛋白的结合电泳条带。第1泳道是游离DNA自身的条带，属于阴性对照；而2号泳道描述的是dCas9和游离DNA(SEQ ID NO.7)结合之后的情况。在阴性对照中，游离DNA的条带清晰完整，电泳成功，而第2条泳道由于游离DNA和dCas9相结合，导致DNA无法正常进行电泳，几乎所有DNA都留在了泳道井中，故而在相对应处没有明显条带。为了计算dCas9和DNA的结合比例(E1)，我们使用以下公式进行计算：
Figure PCTCN2018125973-appb-000004
该公式将1号泳道条带亮度作为总DNA量的基准，2号泳道相对应位置条带亮度为未与dCas9结合的DNA量，于是分子计算后便是与dCas9蛋白结合的DNA的量，再除以总DNA量得出dCas9和DNA的结合效率，计算得到dCas9蛋白和DNA结合效率为97.4％。使用相同的实验和计算方法，表3总结了不同游离DNA长度所对应的dCas9-DNA结合比例，结果显示dCas9能无差别高效的结合上述11种DNA，展现出良好的从溶液中分离核酸的潜质。
表3：各个DNA对应的dCas9-DNA结合比例
Figure PCTCN2018125973-appb-000005
实施例5：偶联了FLAG的dCas9蛋白在溶液中结合游离DNA后，与固相珠结合并将其分离出来
反应体系：
总体积：100μl
dCas9蛋白：10μg
DNA(SEQ ID NO.7、8、9、11，代表不同游离DNA的长度)：每种DNA各0.25μg
无核酸酶双蒸水：定容到100μl
上述反应体系加入1.5ml PCR反应管中混合均匀后，室温或37℃反应15分钟到1小时。
取15μl anti-FLAG亲和凝胶珠(Bimake,B23101)加入1.5毫升离心管中，加入150μl实施例4中的缓冲液(BA)，混合均匀后以5000RPM的转速离心30秒，弃上清，重复上述步骤一次。弃上清后将上述100μl反应体系加入清洗好的亲和凝胶珠中，室温中反转混匀，反应1小时。
反应结束后以5000RPM的转速离心30秒，弃上清，加入150μl实施例4中的缓冲液(BA)，混合均匀后以5000RPM的转速离心30秒，弃上清，重复上述步骤一次。弃上清后在凝胶珠中加入30μl蛋白酶K(天根生化，RT403)，并加入20μl BA。混合均匀后55℃水浴加热30分钟，以5000RPM的转速离心30秒，取上清得到分离出来的游离DNA。经电泳跑胶并与阳性对照(0.25μg DNA溶于30μl蛋白酶K加20μl BA溶液中)比较之后，根据以下公式计算出DNA分离效率E2，并总结在表4中：
Figure PCTCN2018125973-appb-000006
表4：各个DNA对应的dCas9-DNA分离效率
Figure PCTCN2018125973-appb-000007
实施例6：偶联了FLAG的dCas9蛋白与固相珠结合后，在溶液中结合游离DNA并将其分离出来
第一步：取15μl Anti-FLAG亲和凝胶珠(GenScript,L00439-1)加入1.5毫升离心管中，加入150μl实施例4中的缓冲液(BA)，混合均匀后以5000RPM的转速离心30秒，弃上清，重复上述步骤一次。弃上清后在凝胶珠中加入500μl BA，将凝胶珠混合均匀。然后在混合液中加入1μl浓度为10μg/μl dCas9蛋白溶液，室温中反转混匀，反应1小时。
第二步：反应结束后以5000RPM的转速离心30秒，弃上清，加入150μl实施例4中的缓冲液(BA)，混合均匀后以5000RPM的转速离心30秒，弃上清，重复上述步骤一次。将清洗后的凝胶珠分别加入4管1ml含有不同长度游离DNA溶液中(游离DNA使用了SEQ ID NO.7、8、9、11，分别代表不同游离DNA的长度957、486、166和2655bp)，室温或37℃反转混匀反应15分钟到1小时。
第三步：反应结束后以5000RPM的转速离心30秒，弃上清，加入150μl实施例4中的缓冲液(BA)，混合均匀后以5000RPM的转速离心30秒，弃上清，重复上述步骤一次。弃上清后在凝胶珠中加入30μl蛋白酶K(天根生化，RT403)，并加入20μl BA。混合均匀后55℃水浴加热30分钟，以5000RPM的转速离心30秒，取上清得到分离出来的游离DNA。
为了与凯杰现有的试剂盒进行比较，第二步中的1ml游离DNA溶液，使用
Figure PCTCN2018125973-appb-000008
Circulating Nucleic Acid kit(QIAGEN(凯杰)公司)，根据使用说明书的步骤提取溶液中的游离DNA。
阳性对照：相同量的DNA(游离DNA使用了SEQ ID NO.7、8、9、11，分别代表不同游离DNA的长度957、486、166和2655bp)溶解于30μl蛋白酶K(天根生化，RT403)中，并加入20μl BA。
将本发明抓取的DNA、凯杰试剂盒抓取的DNA和阳性对照一起使用Agilent 2100 High Sensitivity DNA Analysis Kit(Cat.5067-4626)进行DNA的定量分析，所得出的抓取DNA量和抓取百分比结果总结在表4中。提取效率为表4中本发明或者凯杰试剂盒抓取DNA的量和阳性对照DNA的量的比值(即为抓取DNA百分比)。本发明游离DNA提 取效率接近80％，而
Figure PCTCN2018125973-appb-000009
Circulating Nucleic Acid kit只有不到50％，因此本发明将游离DNA的提取效率提高了60％左右。
表4：各长度游离DNA用本发明和
Figure PCTCN2018125973-appb-000010
Circulating Nucleic Acid kit的提取效率对比
Figure PCTCN2018125973-appb-000011
实施例7：带有His-Tag的Cpf1(Cas12a)蛋白与固相珠结合后，在溶液中结合游离DNA并将其分离出来
第一步：带有His-Tag的Cpf1(Cas12a)蛋白(IDT，
Figure PCTCN2018125973-appb-000012
A.s.Cas12a(Cpf1)V3，Cat.No.1081068)与His-Tag分离磁珠(Dynabeads TM His-Tag Isolation and Pulldown，Cat.No.10103D)连接。取300μl Dynabeads放入1.5ml离心管中，加1ml pH＝7.4的PBS，混合均匀，将离心管置于磁性分离器(海狸公司，Cat.No.60201)对应的样品孔中，静置两分钟，使磁珠被吸附聚集于管壁，溶液恢复澄清，小心的吸出上清，重复上述步骤两遍。40μl Cpf1蛋白与Dynabeads混匀，加1ml pH＝7.4的PBS，室温放混匀仪上转动20分钟至2小时，将离心管置于磁性分离器对应的样品孔中，静置两分钟，弃上清，加入500μl实施例4中的缓冲液(BA)，混合均匀后将离心管置于磁性分离器对应的样品孔中，静置两分钟，弃上清，重复上述步骤一次。将清洗后的Dynabeads取10到100μl，加入1ml含有SEQ ID NO.9号DNA的溶液中，室温或37℃反转混匀反应15分钟到6小时。
第二步：反应结束后将装有反应溶液和Dynabeads的离心管置于磁性分离器对应的样品孔中，静置两分钟，弃上清，加入150μl实施例4中的缓冲液(BA)，将离心管置于磁性分离器对应的样品孔中，静置两分钟，弃上清，重复上述步骤一次。弃上清后在Dynabeads中加入30μl蛋白酶K(天根生化，RT403)，并加入20μl BA。混合均匀后55℃水浴加热30分钟，将离心管置于磁性分离器对应的样品孔中，静置两分钟，取上清得到分离出来的游离DNA。
为了与凯杰现有试剂盒进行对比，第三步中的1ml游离DNA溶液，使用
Figure PCTCN2018125973-appb-000013
Circulating Nucleic Acid kit(QIAGEN(凯杰)公司)，根据使用说明书的步骤提取血浆中的游离DNA。
阳性对照：相同量的DNA(游离DNA使用了SEQ ID NO.9，代表游离DNA的长度166bp)溶解于30μl蛋白酶K(天根生化，RT403)中，并加入20μl BA。
将本发明抓取的DNA、凯杰试剂盒抓取的DNA和阳性对照一起使用Agilent 2100 High Sensitivity DNA Analysis Kit(Cat.5067-4626)进行DNA的定量分析，所得出的抓取DNA量和抓取百分比结果总结在表5中。提取效率为表5中本发明或者凯杰试剂盒抓取DNA的量和阳性对照DNA的量的比值(即为抓取DNA百分比)。本发明游离DNA提取效率接近80％，而
Figure PCTCN2018125973-appb-000014
Circulating Nucleic Acid kit只有51％，因此本发明将在血浆中提取游离DNA的提取效率提高了54.7％。
表5：160bp游离DNA用本发明和
Figure PCTCN2018125973-appb-000015
Circulating Nucleic Acid kit的提取效率对比
Figure PCTCN2018125973-appb-000016
实施例8：偶联了AVI-Tag的dCas9蛋白与固相珠结合后，在血浆中结合外源游离DNA并将其分离出来
第一步：his-S.P.dCas9-AVI蛋白的纯化，参考实施例3，将FLAG序列替换为AVI-Tag序列(DNA序列：ggcctgaacgatatttttgaagcgcagaaaattgaatggcatgaa)。
第二步：使用AViDITY公司的BirA-500kit(Cat.BirA500)，根据试剂盒的说明书将AVI-Tag-dCas9蛋白生物素化。在1.5ml的离心管中加入相应的反应物进行反应，反应体系如下：
10×BiomixA  8μl
10×BiomixB  8μl
AVI-Tag-dCas9蛋白  50μl
BirA酶  0.8μl
超纯水  补充总体积至80μl
之后将反应液放室温反应1小时或过夜。
第三步：生物素化的蛋白与Streptavidin凝胶珠连接。取400μl GenScript Streptavidin凝胶珠(Cat.No.L00353)放1.5ml离心管中，加1ml pH＝7.4的PBS，室温5000g离心30s，取出后小心的吸出上清，重复上述步骤两遍。80μl生物素化的蛋白与Streptavidin凝胶珠混匀，加1ml pH＝7.4的PBS，室温放混匀仪上转动20分钟至2小时，室温5000g离心30秒，弃上清，加入500μl实施例4中的缓冲液(BA)，混合均匀后以5000RPM的转速离心30秒，弃上清，重复上述步骤一次。将清洗后的凝胶珠取10到100μl，加入1ml含有SEQ ID NO.9号DNA的血浆中，室温或37℃反转混匀反应15分钟到6小时。
第四步：反应结束后以5000RPM的转速离心30秒，弃上清，加入150μl实施例4中的缓冲液(BA)，混合均匀后以5000RPM的转速离心30秒，弃上清，重复上述步骤一次。弃上清后在凝胶珠中加入30μl蛋白酶K(天根生化，RT403)，并加入20μl BA。混合均匀后55℃水浴加热30分钟，以5000RPM的转速离心30秒，取上清得到分离出来的游离DNA。
为了与凯杰现有试剂盒进行对比，第三步中的1ml游离DNA溶液，使用
Figure PCTCN2018125973-appb-000017
Circulating Nucleic Acid kit(QIAGEN(凯杰)公司)，根据使用说明书的步骤提取血浆中的游离DNA。
阳性对照：相同量的DNA(游离DNA使用了SEQ ID NO.9，代表游离DNA的长度166bp)溶解于30μl蛋白酶K(天根生化，RT403)中，并加入20μl BA。
将本发明抓取的DNA、凯杰试剂盒抓取的DNA和阳性对照一起使用Agilent 2100 High Sensitivity DNA Analysis Kit(Cat.5067-4626)进行DNA的定量分析，所得出的抓取DNA量和抓取百分比结果总结在表6中。提取效率为表6中本发明或者凯杰试剂盒抓取DNA的量和阳性对照DNA的量的比值(即为抓取DNA百分比)。本发明游离DNA提取效率接近78％，而
Figure PCTCN2018125973-appb-000018
Circulating Nucleic Acid kit只有51％，因此本发明将在血浆中提取游离DNA的提取效率提高了53％。
表6：160bp游离DNA用本发明和
Figure PCTCN2018125973-appb-000019
Circulating Nucleic Acid kit的提取效率对比
Figure PCTCN2018125973-appb-000020
实施例9：带有His-Tag的Cas9蛋白与固相珠结合后，在血浆中结合外源游离DNA并将其分离出来
第一步：带有His-Tag的Cas9蛋白(IDT，
Figure PCTCN2018125973-appb-000021
S.p.Cas9Nuclease V3，Cat.No.1081058)与His-Tag分离磁珠(Dynabeads TM His-Tag Isolation and Pulldown，Cat.No.10103D)连接。取300μl Dynabeads放入1.5ml离心管中，加1ml pH＝7.4的PBS，混合均匀，将离心管置于磁性分离器(海狸公司，Cat.No.60201)对应的样品孔中，静置两分钟，使磁珠被吸附聚集于管壁，溶液恢复澄清，小心的吸出上清，重复上述步骤两遍。40μlCas9蛋白与Dynabeads混匀，加1ml pH＝7.4的PBS，室温放混匀仪上转动20分钟至2小时，将离心管置于磁性分离器对应的样品孔中，静置两分钟，弃上清，加入500μl实施例4中的缓冲液(BA)，混合均匀后将离心管置于磁性分离器对应的样品孔中，静置两分钟，弃上清，重复上述步骤一次。将清洗后的Dynabeads取10到100μl，加入1ml含有SEQ ID NO.9号DNA的血浆中，室温或37℃反转混匀反应15分钟到6小时。
第二步：反应结束后将含有血浆和Dynabeads的离心管置于磁性分离器对应的样品孔中，静置两分钟，弃上清，加入150μl实施例4中的缓冲液(BA)，将离心管置于磁性分离器对应的样品孔中，静置两分钟，弃上清，重复上述步骤一次。弃上清后在Dynabeads中加入30μl蛋白酶K(天根生化，RT403)，并加入20μl BA。混合均匀后55℃水浴加热30分钟，将离心管置于磁性分离器对应的样品孔中，静置两分钟，取上清得到分离出来的游离DNA。
为了与凯杰现有试剂盒进行对比，第三步中的1ml游离DNA溶液，使用
Figure PCTCN2018125973-appb-000022
Circulating Nucleic Acid kit(QIAGEN(凯杰)公司)，根据使用说明书的步骤提取血浆中的游离DNA。
阳性对照：相同量的DNA(游离DNA使用了SEQ ID NO.9，代表游离DNA的长度166bp)溶解于30μl蛋白酶K(天根生化，RT403)中，并加入20μl BA。
将本发明抓取的DNA、凯杰试剂盒抓取的DNA和阳性对照一起使用Agilent 2100 High Sensitivity DNA Analysis Kit(Cat.5067-4626)进行DNA的定量分析，所得出的抓取DNA量和抓取百分比结果总结在表7中。提取效率为表7中本发明或者凯杰试剂盒抓取DNA的量和阳性对照DNA的量的比值(即为抓取DNA百分比)。本发明游离DNA提 取效率超过85％，而
Figure PCTCN2018125973-appb-000023
Circulating Nucleic Acid kit只有55％，因此本发明将在血浆中提取游离DNA的提取效率提高了54.5％。
表7：160bp游离DNA用本发明和
Figure PCTCN2018125973-appb-000024
Circulating Nucleic Acid kit的提取效率对比
Figure PCTCN2018125973-appb-000025
实施例10：偶联了AVI-Tag的dCas9蛋白与固相珠结合后，在血浆中结合内源游离DNA并将其分离出来
第一步与第二步与实施例8中的第一步和第二步相同，得到生物素化的dCas9蛋白。
第三步：生物素化的蛋白与Streptavidin凝胶珠连接。取400μl GenScript Streptavidin凝胶珠(Cat.No.L00353)放1.5ml离心管中，加1ml pH＝7.4的PBS，室温5000g离心30s，取出后小心的吸出上清，重复上述步骤两遍。80μl生物素化的蛋白与Streptavidin凝胶珠混匀，加1ml pH＝7.4的PBS，室温放混匀仪上转动20分钟至2小时，室温5000g离心30秒，弃上清，加入500μl实施例4中的缓冲液(BA)，混合均匀后以5000RPM的转速离心30秒，弃上清，重复上述步骤一次。将清洗后的凝胶珠取10到100μl，加入1ml血浆中，室温或37℃反转混匀反应15分钟到6小时。
第四步：反应结束后以5000RPM的转速离心30秒，弃上清，加入150μl实施例4中的缓冲液(BA)，混合均匀后以5000RPM的转速离心30秒，弃上清，重复上述步骤一次。弃上清后在凝胶珠中加入30μl蛋白酶K(天根生化，RT403)，并加入20μl BA。混合均匀后55℃水浴加热30分钟，以5000RPM的转速离心30秒，取上清得到分离出来的游离DNA。
为了与凯杰现有试剂盒进行对比，第三步中的1ml游离DNA溶液，使用
Figure PCTCN2018125973-appb-000026
Circulating Nucleic Acid kit(QIAGEN(凯杰)公司)，根据使用说明书的步骤提取血浆中的游离DNA。
将本发明抓取的DNA和凯杰试剂盒抓取的DNA一起使用Agilent 2100 High Sensitivity DNA Analysis Kit(Cat.5067-4626)进行DNA的定量分析，所得出的抓取DNA量结果总结在表8中。本发明游离DNA提取量接近28.49pg/μl，而
Figure PCTCN2018125973-appb-000027
Circulating Nucleic Acid kit只有24.68pg/μl，因此本发明将在血浆中提取内源游离DNA的提取效率提高了15.4％。
表8：血浆游离DNA用本发明和
Figure PCTCN2018125973-appb-000028
Circulating Nucleic Acid kit的提取量对比
Figure PCTCN2018125973-appb-000029

Claims (33)

  1. 一种采用Cas蛋白从DNA溶液中分离DNA的方法。
  2. 权利要求1所述的方法，包括步骤a)DNA溶液与Cas蛋白体系混合；c)从Cas蛋白体系与DNA结合的复合物中分离得到DNA。
  3. 权利要求1所述的方法，包括步骤a)DNA溶液与Cas蛋白体系混合；b)富集步骤a)中的Cas蛋白体系与DNA结合的复合物；c)从步骤b)富集的复合物中分离得到DNA。
  4. 权利要求2所述的方法，其中所述的Cas蛋白体系为偶联了平板状、膜状或柱状的固体支持物的Cas蛋白。
  5. 权利要求3所述的方法，其中所述的Cas蛋白体系为偶联了珠形固体支持物的Cas蛋白。
  6. 权利要求3所述的方法，其中所述的Cas蛋白体系为Cas蛋白与至少一种连接序列形成的Cas融合蛋白。
  7. 权利要求6所述的方法，其中步骤b)在溶液中加入偶联了亲和分子的固体支持物，再通过离心富集或磁性富集。
  8. 权利要求6所述的方法，其中在步骤a)中加入偶联了亲和分子的固体支持物，孵育后，再通过步骤b)富集。
  9. 权利要求1-8任一项所述的方法，其中所述的DNA是双链DNA。
  10. 权利要求4或5所述的方法，其中所述的Cas蛋白体系为Cas蛋白通过化学共价键偶联到固体支持物。
  11. 权利要求6所述的方法，其中所述的Cas蛋白体系为Cas融合蛋白与固体支持物上的亲和分子结合而形成的。
  12. 权利要求4-11任一项所述的方法，其中所述的固体支持物是能与氨基酸残基形成化学键的固体物质。
  13. 权利要求10所述的方法，其中所述的固体支持物选自凝胶材料、磁性材料、纤维素材料、硅胶材料、玻璃材料和人造高分子聚合物制得的固体物质中的至少一种。
  14. 权利要求5-13任一项所述的方法，其中所述的珠形的固体支持物为磁珠、凝胶珠和/或硅胶珠。
  15. 权利要求6-14任一项所述的方法，其中所述的连接序列和亲和分子选自下组配体和受体组合中的一种：酶与底物、抗原与抗体、生物素与亲和素。
  16. 权利要求15所述的方法，其中所述的酶与底物选自谷胱甘肽转移酶与谷胱甘肽。
  17. 权利要求15所述的方法，其中所述的抗原-抗体选自下组中的一种：A蛋白或其保留了结合Fc区域功能性的片段与免疫球蛋白或其Fc或Fab片段、G蛋白或其保留了结合Fc区域功能性的片段与免疫球蛋白或其Fc或Fab片段、组氨酸标签与抗组胺酸标签、多组氨酸标签与抗多组氨酸标签和FLAG标签与抗FLAG标签。
  18. 权利要求15所述的方法，其中所述的生物素与亲和素选自下组中的一种：生物素与亲和素或链酶亲和素、连接了生物素的生物素受体蛋白与亲和素或链酶亲和素、Strep-tag与亲和素或链酶亲和素。
  19. 权利要求2-18任一项所述的方法，其中所述的步骤a)的反应时间为5分钟到6小时，优选反应时间为15分钟到1小时。
  20. 权利要求2-18任一项所述的方法，其中所述的步骤a)的反应温度为20-37℃，优选反应温度为30-37℃。
  21. 权利要求7、12-20任一项所述的方法，其中所述的步骤b)，在溶液中加入偶联了亲和分子的固体支持物的反应时间为30分钟至过夜，优选反应时间为1-2小时。
  22. 权利要求21所述的方法，其中所述的步骤b)的反应温度为4℃到37℃，优选反应温度为20-30℃。
  23. 权利要求3-22任一项所述的方法，其中所述b)的富集为离心富集或磁性富集。
  24. 权利要求2-23任一项所述的方法，其中所述步骤c)通过洗脱分离DNA。
  25. 权利要求2-24任一项所述的方法，其中所述的Cas是具有DNA结合功能的Cas蛋白。
  26. 权利要求25所述的方法，其中所述的Cas蛋白是天然的Cas蛋白、突变后失去了gRNA结合能力的Cas蛋白，或者突变后核酸酶失活的Cas蛋白。
  27. 权利要求25或26所述的方法，其中Cas蛋白选自Cas1、Cas1B、Cas2、Cas3、Cas4、Cas5、Cas6、Cas7、Cas8、Cas9、Cas10、Cas12、Cas13和Cas14。
  28. 权利要求27所述的方法，其中所述的Cas蛋白是Cas9蛋白。
  29. 权利要求28所述的方法，其中所述的Cas9蛋白是dCas9蛋白(突变后核酸酶失活的Cas9蛋白)。
  30. 一种从溶液中分离DNA的试剂盒，包含组分a)Cas蛋白体系，和组分b)DNA洗脱液。
  31. 权利要求29所述的试剂盒，其中组分a)Cas蛋白体系是偶联了固体支持物的Cas蛋白溶液体系，或是含有Cas融合蛋白和偶联了亲和分子的固体支持物的溶液体系。
  32. 权利要求30所述的试剂盒，其中组分a)Cas蛋白体系由两个独立的体系组成，i)Cas蛋白与至少一种连接序列形成的Cas融合蛋白，和ii)偶联了亲和分子的固体支持物。
  33. 权利要求30所述的试剂盒，其中组分b)DNA洗脱液可以是以下液体：0.5-10mg/ml蛋白酶K溶液、1-5M浓度的NaCl或者KCl盐溶液、具有0-0.3M Tris和0-0.5M NaCl的pH 11-12.5的碱洗脱液。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110791578A (zh) * 2019-11-28 2020-02-14 广州微远基因科技有限公司 用于百日咳博德特氏杆菌的crispr检测引物组及其用途

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022210822A1 (ja) * 2021-04-02 2022-10-06 キヤノン株式会社 試薬、及びそれを用いた標的核酸の検出方法
CN113249376A (zh) * 2021-05-18 2021-08-13 明科生物技术(杭州)有限公司 一种基于CRISPR-Cas捕获甜瓜线粒体基因组的方法及该方法的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015120445A1 (en) 2014-02-10 2015-08-13 Nancy Quan Isolation of cell-free nucleic acids from bodily fluid samples using solid chaotropic agents
WO2016198571A1 (en) 2015-06-10 2016-12-15 Qiagen Gmbh Method for isolating extracellular nucleic acids using anion exchange particles
CN106978428A (zh) * 2017-03-15 2017-07-25 上海吐露港生物科技有限公司 一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及试剂盒
CN107406875A (zh) * 2014-12-20 2017-11-28 阿克生物公司 使用CRISPR/Cas系统蛋白靶向消减、富集、和分割核酸的组合物及方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9873907B2 (en) * 2013-05-29 2018-01-23 Agilent Technologies, Inc. Method for fragmenting genomic DNA using CAS9

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015120445A1 (en) 2014-02-10 2015-08-13 Nancy Quan Isolation of cell-free nucleic acids from bodily fluid samples using solid chaotropic agents
CN107406875A (zh) * 2014-12-20 2017-11-28 阿克生物公司 使用CRISPR/Cas系统蛋白靶向消减、富集、和分割核酸的组合物及方法
WO2016198571A1 (en) 2015-06-10 2016-12-15 Qiagen Gmbh Method for isolating extracellular nucleic acids using anion exchange particles
CN106978428A (zh) * 2017-03-15 2017-07-25 上海吐露港生物科技有限公司 一种Cas蛋白特异结合靶标DNA、调控靶标基因转录的方法及试剂盒

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
CHRISTOPHER A. V ET AL., NATURE MEDICINE, vol. 24, 2018, pages 1216 - 1224
CONG ET AL., SCIENCE, vol. 339, 2013, pages 819 - 823
FUJITA, T. ET AL.: "Efficient Sequence-specific Isolation of DNA Fragments and Chromatin by in Vitro enChIP Technology Using Recombinant CRISPR Ribonucleoproteins", GENES TO CELLS, vol. 21, no. 4, 31 December 2016 (2016-12-31), pages 370 - 377, XP055621734 *
HERMANSON ET AL.: "Immobilized Affinity Ligand Techniques", 1992, ACADEMIC PRESS
JANA M ET AL., PLANT BIOTECHNOLOGY JOURNAL, vol. 15, 2017, pages 917 - 926
JINEK ET AL., SCIENCE, vol. 337, 2012, pages 816 - 821
JOHNNY H. H ET AL., NATURE, vol. 556, no. 7699, 2018, pages 57 - 63
KOMOR ET AL., SCI. ADV., vol. 3, 2017, pages eaao4774
MA ET AL., ACS SYNTH. BIOL., vol. 7, no. 4, 2018, pages 978 - 985
MANDEL P., C R HEBD SEANCES ACAD SCI (PARIS, vol. 142, no. 3, 1948, pages 241 - 3
QI ET AL., CELL, vol. 152, 2013, pages 1173 - 1183
XUE ET AL., CLINICA CHIMICA ACTA, vol. 404, 2009, pages 100 - 104

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110791578A (zh) * 2019-11-28 2020-02-14 广州微远基因科技有限公司 用于百日咳博德特氏杆菌的crispr检测引物组及其用途

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